Fish Allergy Antigen

Abstract

The present invention provides novel antigens of an allergy to fish, methods and kits for diagnosing an allergy to fish, a pharmaceutical composition comprising the antigen, fish or fish eggs in which the antigen is eliminated, a processed product of the fish or the fish eggs or fish which lay the fish eggs or have hatched from the eggs, and a tester for determining the presence or absence of a fish antigen in an object of interest.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel antigen of an allergy to fish. The present invention also relates to a kit, a composition, and a method for diagnosing allergy to fish. The present invention also relates to a pharmaceutical composition comprising the antigen and fish or fish eggs in which the antigen is eliminated or reduced, a processed product of the fish or the fish eggs or fish which lay the fish eggs or have hatched from the eggs. The present invention further relates to a tester for determining the presence or absence of a fish antigen in an object of interest.

BACKGROUND ART

[0002] In blood and tissues of allergic patients, IgE antibodies specific to particular antigens are produced. Physiological consequences caused by interaction between such IgE antibodies and such particular antigens elicit allergic reactions.

[0003] In the process of production of conventional allergy testing agents, antigen reagents are commonly prepared simply by grinding a candidate allergenic food, food material or the like (Patent Literature 1). Therefore, numerous proteins were contained in a reagent and there the content of each protein was very little. For this reason, the only case where conventional allergy tests have permitted detection of a positive allergic reaction is when in a conventional antigen reagent containing many proteins, a particular antigen protein which causes the allergic reaction (allergen component) is present in an amount exceeding a threshold that allows determination of a positive reaction for binding to an IgE antibody. However, no determination of a positive reaction was possible and diagnosis efficiency was not sufficiently high when using a conventional allergy testing agent in patients possessing an IgE antibody binding to an allergen component present in small amounts in an allergen such as food, food material or the like.

[0004] The severity and symptoms of an allergic reaction do not necessarily correlate with the content of an allergen component. Even when a patient's IgE antibody reacts with an allergen component present in trace amounts in a candidate allergic food and food material, the allergic reaction may develop allergic symptoms or may affect the severity of those symptoms.

[0005] An attempt to increase the diagnostic efficiency is being made by examining IgE antibodies to each protein composing food and food material to distinguish sensitization that directly contributes to a diagnosis from sensitization based on cross-antigenicity by a pan-allergen or the like. The allergens set forth in Table below are currently known as fish allergens (Non Patent Literature 1).

[0006] However, while it is necessary to exhaustively identify allergen components in candidate allergic foods and food materials in order to enhance the reliability of allergy tests, the patient detection rate by the measurement of such allergen components is far from sufficient. Identification of novel allergens in fish is very important not only for increasing the precision of diagnosis, but also for determining targets of therapeutic agents and low allergenic foods and low allergenic food materials.

[0007] Meanwhile, in the field of protein separation and purification, various efforts have conventionally been made to develop methods for separating and purifying a protein or nucleic acid of interest from cell extracts or the like. Such methods may well be exemplified by dialysis based on salt concentration, and centrifugal separation.

[0008] Other efforts have been made to develop many purification methods based on electric charges of protein or nucleic acid residues or on the difference in molecular weight. Electric charge-based purification methods can be exemplified by column chromatography using ion exchange resins, and isoelectric focusing. Purifications based on molecular weight difference can be exemplified by centrifugal separation, molecular-sieve column chromatography, and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

[0009] In recent years, a method for separating and purifying many different proteins from a small amount of sample has been used, which is more specifically a two-dimensional electrophoresis consisting of isoelectric focusing in the first dimension, followed by SDS-PAGE in the second dimension. The present applicant has conventionally developed some 2D electrophoresis methods with high separation ability (Patent Literature 2-5).

CITATION LIST

Patent Literature

[0010] PTL1: Japanese Patent Application Publication No. JP 2002-286716

[0011] PTL2: Japanese Patent Application Publication No. JP 2011-33544

[0012] PTL3: Japanese Patent Application Publication No. JP 2011-33546

[0013] PTL4: Japanese Patent Application Publication No. JP 2011-33547

[0014] PTL5: Japanese Patent Application Publication No. JP 2011-33548

Non-Patent Literature

[0015] NPTL1: Allergen Nomenclature, WHO/IUIS Allergen Nomenclature Sub-Committee, [searched on Jun. 9, 2016], internet<URL: http://www.allergen.org/search.php?allergenname=&allergensource=&TaxSourc- e=Animalia+Chordata&TaxOrder=&foodallerg=1&bioname=>

SUMMARY OF INVENTION

Technical Problem

[0016] The present invention provides novel antigens of an allergy to fish. The present invention also provides methods and kits for diagnosing allergy to fish. The present invention also provides pharmaceutical compositions comprising such an antigen and fish or fish eggs in which such an antigen is eliminated or reduced, a processed product of the fish or the fish eggs or fish which lay the fish eggs or have hatched from the eggs.

Solution to Problem

[0017] In order to solve the aforementioned problems, the present inventors had made intensive studies to identify causative antigens of an allergy to fish. As a result, the inventors succeeded in identifying novel antigens to which an IgE antibody in the serum of a fish-allergic patient specifically binds. The present invention has been completed based on this finding.

[0018] Thus, in one embodiment, the present invention can be as defined below.

[0019] [1] A kit for diagnosing a fish allergy, the kit comprising, as an antigen, at least one protein defined in any one of the following (1) to (9): [0020] (1) (1A) Alpha-actinin-3 or a variant thereof, which is a protein defined below in any of (1A-a) to (1A-e) which is an antigen for the fish allergy:

[0021] (1A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 2;

[0022] (1A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2;

[0023] (1A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 1;

[0024] (1A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 1; or (1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1; or (1B) a protein comprising an amino acid sequence of SEQ ID NO: 2 or 3; [0025] (2) (2A) EEF1A2 binding protein-like or a variant thereof, which is a protein defined below in any of (2A-a) to (2A-e) which is an antigen for the fish allergy:

[0026] (2A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 5;

[0027] (2A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 5;

[0028] (2A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 4;

[0029] (2A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 4; or

[0030] (2A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4; or

[0031] (2B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8; [0032] (3) (3A) Alpha-1,4-glucan phosphorylase or a variant thereof, which is a protein defined below in any of (3A-a) to (3A-e) which is an antigen for the fish allergy:

[0033] (3A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 10;

[0034] (3A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 10;

[0035] (3A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 9;

[0036] (3A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 9; or

[0037] (3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9; or

[0038] (3B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10-16; [0039] (4) (4A) Elongation factor 2 or a variant thereof, which is a protein defined below in any of (4A-a) to (4A-e) which is an antigen for the fish allergy:

[0040] (4A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 19;

[0041] (4A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 19;

[0042] (4A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 18;

[0043] (4A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 18; or

[0044] (4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 18; or

[0045] (4B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; [0046] (5) (5A) Heat shock cognate 70 kDa protein or a variant thereof, which is a protein defined below in any of (5A-a) to (5A-e) which is an antigen for the fish allergy:

[0047] (5A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 26;

[0048] (5A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 26;

[0049] (5A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 25;

[0050] (5A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 25; or

[0051] (5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 25; or

[0052] (5B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31; [0053] (6) (6A) Serotransferrin or a variant thereof, which is a protein defined below in any of (6A-a) to (6A-e) which is an antigen for the fish allergy:

[0054] (6A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 33;

[0055] (6A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 33;

[0056] (6A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 32;

[0057] (6A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 32; or

[0058] (6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 32; or

[0059] (6B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 33-41; [0060] (7) (7A) Myosin binding protein H-like or a variant thereof, which is a protein defined below in any of (7A-a) to (7A-e) which is an antigen for the fish allergy:

[0061] (7A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 43;

[0062] (7A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 43;

[0063] (7A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 42;

[0064] (7A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 42; or

[0065] (7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 42; or

[0066] (7B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 43-54, [0067] (8) (8A) Desmin (Fragment) or a variant thereof, which is a protein defined below in any of (8A-a) to (8A-e) which is an antigen for the fish allergy:

[0068] (8A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 56;

[0069] (8A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 56;

[0070] (8A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 55;

[0071] (8A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 55; or

[0072] (8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 55; or

[0073] (8B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59; [0074] (9) (9A) Capping protein (Actin filament) muscle Z-line beta or a variant thereof, which is a protein defined below in any of (9A-a) to (9A-e) which is an antigen for the fish allergy:

[0075] (9A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 61;

[0076] (9A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 61;

[0077] (9A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 60;

[0078] (9A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 60; or

[0079] (9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 60; or

[0080] (9B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 61-68.

[0081] [2] A composition for diagnosing a fish allergy, the composition comprising, as an antigen, at least one of proteins as defined above in any of (1) to (9) of [1].

[0082] [3] A method for providing an indicator for diagnosing a fish allergy in a subject, the method comprising the steps of: [0083] (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody; [0084] (ii) detecting binding between an IgE antibody present in the sample from the subject and the antigen; and [0085] (iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject has a fish allergy is provided; [0086] wherein the antigen is at least one of proteins as defined above in any of (1) to (9) of [1].

[0087] [4] A pharmaceutical composition comprising at least one of proteins as defined above in any of (1) to (9) of [1].

[0088] [5] The pharmaceutical composition as set forth in [4] for the treatment of a fish allergy.

[0089] [6] A fish or a processed product of fish, in which an antigen is eliminated or reduced, wherein the antigen is at least one of proteins as defined above in any of (1) to (9) of [1].

[0090] [7] A tester for determining the presence or absence of a fish antigen in an object of interest, the tester comprising an antibody that binds to at least one of proteins as defined above in any of (1) to (9) of [1].

[0091] [8] A tester for determining the presence or absence of an antigen causing an allergy to fish in an object of interest, the tester comprising a primer having a nucleotide sequence complementary to at least one part of one of the nucleotide sequences set forth in SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, or 60.

[0092] [9] A kit for diagnosing a fish allergy, the kit comprising, as an antigen, at least one protein defined in any one of the following (10) to (14): [0093] (10) (10A) Myosin heavy chain, fast skeletal muscle-like or a variant thereof, which is a protein defined below in any of (10A-a) to (10A-e) which is an antigen for the fish allergy:

[0094] (10A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 70;

[0095] (10A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 70;

[0096] (10A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 69;

[0097] (10A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 69; or

[0098] (10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 69; or

[0099] (10B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 70-108; [0100] (11) (11A) Glycogen phosphorylase, muscle form-like or a variant thereof, which is a protein defined below in any of (11A-a) to (11A-e) which is an antigen for the fish allergy:

[0101] (11A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 110;

[0102] (11A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 110;

[0103] (11A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 109;

[0104] (11A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 109; or

[0105] (11A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 109; or

[0106] (11B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 110-119; [0107] (12) (12A) Myosin-binding protein C, fast-type-like or a variant thereof, which is a protein defined below in any of (12A-a) to (12A-e) which is an antigen for the fish allergy:

[0108] (12A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 121;

[0109] (12A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 121;

[0110] (12A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 120;

[0111] (12A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 120; or

[0112] (12A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 120; or

[0113] (12B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 121-136; [0114] (13) (13A) ATP synthase subunit beta, mitochondrial or a variant thereof, which is a protein defined below in any of (13A-a) to (13A-e) which is an antigen for the fish allergy:

[0115] (13A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 138;

[0116] (13A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 138;

[0117] (13A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 137;

[0118] (13A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 137; or

[0119] (13A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 137; or

[0120] (13B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 138-142; [0121] (14) (14A) L-lactate dehydrogenase A chain-like or a variant thereof, which is a protein defined below in any of (14A-a) to (l4A-e) which is an antigen for the fish allergy:

[0122] (14A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 144;

[0123] (14A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 144;

[0124] (14A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 143;

[0125] (14A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 143; or

[0126] (14A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 143; or

[0127] (14B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144-149.

[0128] [10] A composition for diagnosing a fish allergy, the composition comprising, as an antigen, at least one of proteins as defined above in any of (10) to (14) of [9].

[0129] [11] A method for providing an indicator for diagnosing a fish allergy in a subject, the method comprising the steps of: [0130] (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody; [0131] (ii) detecting binding between an IgE antibody present in the sample from the subject and the antigen; and [0132] (iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject has a fish allergy is provided; [0133] wherein the antigen is at least one of proteins as defined above in any of (10) to (14) of [9].

[0134] [12] A pharmaceutical composition comprising at least one of proteins as defined above in any of (10) to (14) of [9].

[0135] [13] The pharmaceutical composition as set forth in [12] for the treatment of a fish allergy.

[0136] [14] A fish or a processed product of fish, in which an antigen is eliminated or reduced, wherein the antigen is at least one of proteins as defined above in any of (10) to (14) of [9].

[0137] [15] A tester for determining the presence or absence of a fish antigen in an object of interest, the tester comprising an antibody that binds to at least one of proteins as defined above in any of (10) to (14) of [9].

[0138] [16] A tester for determining the presence or absence of an antigen causing an allergy to fish in an object of interest, the tester comprising a primer having a nucleotide sequence complementary to at least one part of one of the nucleotide sequences set forth in SEQ ID NO: 69, 109, 120, 137, or 143.

[0139] [17] A fish-derived antigen which is at least one of proteins as defined above in any of (1) to (9) of [1] and causes an allergy to fish.

[0140] [18] A fish-derived antigen which is at least one of proteins as defined above in any of (10) to (14) of [9] and causes an allergy to fish.

Advantageous Effects of Invention

[0141] The present invention can provide novel antigens of an allergy to fish. Since the novel antigens (allergen components) that trigger a fish allergy were identified in the present invention, it is possible to provide highly sensitive methods and kits for diagnosing an allergy to fish, pharmaceutical compositions comprising such an antigen, fish or fish eggs in which the antigen is eliminated or reduced, a processed product of the fish or the fish eggs or fish which lay the fish eggs or have hatched from the eggs, and testers for determining the presence or absence of a fish antigen in an object of interest.

BRIEF DESCRIPTION OF DRAWINGS

[0142] FIG. 1A is a photograph of a gel showing a protein electrophoretic pattern in two-dimensional electrophoresis of proteins contained in an extract of flesh of salmon (left panel) and a photograph of an immunoblot obtained from the two-dimensional electrophoretic pattern by using a serum of Fish-allergic patient 1 (right panel). The bands at the left of the photograph of the gel are bands of molecular weight markers and the numbers on the left side of the photograph of the gel are molecular weights of respective molecular weight markers (KDa). The numbers on the upper side of the photograph indicate isoelectric points.

[0143] FIG. 1B is a photograph of an immunoblot obtained from the two-dimensional electrophoretic pattern of the proteins contained in the extract of the flesh of salmon by using a serum of Fish-allergic patient 2 (left panel) and a photograph of an immunoblot of the proteins stained with serum of Fish-allergic patient 3 (right panel). The numbers on the upper side of each photograph indicate isoelectric points.

[0144] FIG. 2 is photographs of immunoblots obtained from two-dimensional electrophoretic patterns of proteins contained in extracts of the flesh of 10 species of fish stained with serum of Fish-allergic patient 1.

[0145] FIG. 3 is photographs of immunoblots obtained from two-dimensional electrophoretic patterns of proteins contained in extracts of the flesh of 10 species of fish stained with serum of a subject who does not exhibit fish allergic symptoms (Non-fish-allergic subject).

[0146] FIG. 4 is photographs of immunoblots obtained from two-dimensional electrophoretic patterns of proteins contained in extracts of the fish of 6 species of fish by using a serum of Fish-allergic patient 4.

DESCRIPTION OF EMBODIMENTS

[0147] The present invention will be described in detail below, but the present invention is not limited to them.

[0148] Unless otherwise defined herein, all scientific and technical terms used in relation to the present invention shall have meanings commonly understood by those skilled in the art.

[0149] As referred to herein, the "allergy" refers to the state in which, when a certain antigen reenters a living organism sensitized to said antigen, the living organism shows a hypersensitive reaction detrimental to the living organism. In blood and tissues of individuals with many food-allergic diseases, IgE antibodies specific to antigens are produced. IgE antibodies bind to mast cells or basophils. When an antigen specific to such an IgE antibody reenters the body of a patient with an allergic disease, said antigen combines with the IgE antibody bound to mast cells or basophils, and the IgE antibody crosslinks said antigen on the cell surface, resulting in physiological effects of IgE antibody-antigen interaction. Examples of such physiological effects include release of histamine, serotonin, heparin, eosinophil chemotactic factors, leucotrienes, or the like. These released substances provoke an allergic reaction resulting from the combination of an IgE antibody with particular antigens. Such allergic reactions caused by particular antigens occur through the aforementioned pathway.

[0150] As used herein, the term fish means teleost and elasmobranch among fishes, preferably teleost, more preferably those belonging to Salmoniformes, Perciformes, Anguilliformes, Gadiformes, and Pleuronectiformes, further preferably those belonging to Salmonidae, Carangidae, Congridae, Sparidae, Scombridae, Gadidae, Anguillidae, and Paralichthyidae, and even preferably salmon, horse mackerel, sea eel, black porgy, mackerel, sea bream, cod, young yellowtail, eel, and flatfish. The fish may be edible.

[0151] As used herein, the term fish eggs means eggs of fish. The fish eggs may be edible.

[0152] As referred to herein, the "allergy to fish" refers to the state in which an individual has an allergic reaction caused by proteins, etc. present in fish which act as an antigen. The allergy to fish can produce an allergic reaction upon contact with, or consumption of, an antigen present in fish. In general, allergic reactions caused by consumption of foods are particularly referred to as "food allergies". The allergy to fish may be a food allergy.

[0153] As referred to herein, the "antigen" refers to a substance that provokes an allergic reaction, and is also referred to as an "allergen component". The antigen is preferably a protein.

[0154] As referred to herein, the "protein" refers to a molecule having a structure in which naturally occurring amino acids are joined together by peptide bond. The number of amino acids present in a protein is not particularly limited, but proteins having about 2 to 50 amino acids joined together by peptide bond are in some cases called "peptides". In the case where amino acids can form different enantiomers, the amino acids are understood to form an L-enantiomer, unless otherwise indicated. The amino acid sequences of proteins or peptides as used herein are represented by one-letter symbols of amino acids in accordance with standard usage and the notational convention commonly used in the art. The leftward direction represents the amino-terminal direction, and the rightward direction represents the carboxy-terminal direction.

[0155] Identification of Antigens

[0156] Proteins contained in fish were analyzed by the aforementioned technique to identify antigens of an allergy to fish. To be specific, proteins were extracted from the flesh of fish and subjected to two-dimensional electrophoresis under the conditions described below.

[0157] The electrophoresis in the first dimension was isoelectric focusing, which was performed using isoelectric focusing gels with a gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10. The pH gradient of the gels in the direction of electrophoresis was as follows: when the total gel-strip length is taken as 1, the gel-strip length up to pH 5 is taken as "a", the gel-strip length from pH 5 to 7 is taken as "b", and the gel-strip length above pH 7 is taken as "c", "a" is in the range of 0.15 to 0.3, "b" is in the range of 0.4 to 0.7, and "c" is in the range of 0.15 to 0.3. More specifically, the isoelectric focusing was performed using the IPG gels, Immobiline Drystrip (pH 3-10 NL), produced by GE Healthcare Bio-Sciences Corporation (hereinafter abbreviated as "GE"). The electrophoresis system used was IPGphor produced by GE. The maximum current of the electrophoresis system was limited to 75 .mu.A per gel strip. The voltage program adopted to perform the first-dimensional isoelectric focusing was as follows: (1) a constant voltage step was performed at a constant voltage of 300 V until the volt-hours reached 750 Vhr (the current variation width during electrophoresis for 30 minutes before the end of this step was 5 .mu.A); (2) the voltage was increased gradually to 1000 V for 300 Vhr; (3) the voltage was further increased gradually to 5000 V for 4500 Vhr; and then (4) the voltage was held at a constant voltage of 5000 V until the total Vhr reached 12000.

[0158] The electrophoresis in the second dimension was SDS-PAGE, which was performed using polyacrylamide gels whose gel concentration at the distal end in the direction of electrophoresis was set to 3 to 6% and whose gel concentration at the proximal end was set to a higher value than that at the distal end. More specifically, the SDS-PAGE was performed using NuPAGE 4-12% Bris-Tris Gels (IPG well, Mini, 1 mm) produced by Life Technologies. The electrophoresis system used was XCell SureLock Mini-Cell produced by Life Technologies. The electrophoresis was run at a constant voltage of 200 V for about 45 minutes using an electrophoresis buffer composed of 50 mM MOPS, 50 mM Tris base, 0.1% (w/v) SDS and 1 mM EDTA.

[0159] As a result, the following spots in a two-dimensional electrophoresis gel run under the conditions described above for fish proteins have been revealed to exhibit specific binding to IgE antibodies from patients having an allergy to fish and being diagnosed as having immediate-type allergy. [0160] Spot 1: Molecular weight 80 to 160 kDa, pI 3.0 to 7.0 [0161] Spot 2: Molecular weight 110 to 260 kDa, pI 4.0 to 8.0 [0162] Spot 3: Molecular weight 80 to 160 kDa, pI 4.0 to 10.0 [0163] Spot 4: Molecular weight 80 to 160 kDa, pI 5.0 to 11.0 [0164] Spot 5: Molecular weight 50 to 110 kDa, pI 3.0 to 7.0 [0165] Spot 6: Molecular weight 60 to 110 kDa, pI 4.0 to 8.0 [0166] Spot 7: Molecular weight 40 to 80 kDa, pI 4.0 to 8.0 [0167] Spot 8: Molecular weight 40 to 80 kDa, pI 3.0 to 7.0 [0168] Spot 9: Molecular weight 20 to 50 kDa, pI 3.0 to 7.0 [0169] Spot 10: Molecular weight 160 to 300 kDa, pI 3.0 to 7.0 [0170] Spot 11: Molecular weight 80 to 160 kDa, pI 4.0 to 8.0 [0171] Spot 12: Molecular weight 100 to 160 kDa, pI 4.0 to 7.0 [0172] Spot 13: Molecular weight 30 to 70 kDa, pI 3.0 to 7.0 [0173] Spot 14: Molecular weight 20 to 50 kDa, pI 5.0 to 9.0

[0174] Antigen

[0175] (1) Antigen in Spot 1

[0176] As the result of sequence identification of the antigen in spot 1 by mass spectroscopy, the following amino acid sequences of SEQ ID NO: 3 were detected.

[0177] Also, the mass spectroscopic data (SEQ ID NO: 3) obtained for spot 1 on a mass spectrometer was analyzed by comparing the data against the NCB1 protein data, and as a result, the antigen in question was identified as Alpha-actinin-3 (amino acid sequence: SEQ ID NO: 2, encoding nucleotide sequence: SEQ ID NO: 1). SEQ ID NO: 3 corresponds to amino acid 572 to 585 in SEQ ID NO: 2.

[0178] Accordingly, in the present invention, the antigen in spot 1 can be any of (1A-a) to (1A-e) and (1B) as defined below: [0179] (1A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 2; [0180] (1A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 2; [0181] (1A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 1; [0182] (1A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 1; [0183] (1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1; [0184] (1B) a protein comprising at least one amino acid sequence of SEQ ID NO: 2 or 3, preferably a protein comprising all sequences of the amino acid sequences. As referred to above, the amino acid sequence of SEQ ID NO: 2 or 3 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0185] The proteins of (1A-a) to (1A-e) and (1B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0186] The proteins of (1A-a) to (1A-e) and (1B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 80 to 160 kDa, preferably around 90 to 120 kDa and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 3.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0187] Preferably, a protein that is an antigen in spot 1 is an antigen of an allergy to fish.

[0188] (2) Antigen in Spot 2

[0189] As the result of sequence identification of the antigen in spot 2 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 6-8 were detected.

[0190] Also, the mass spectroscopic data obtained for spot 2 (SEQ ID NOs: 6-8) on a mass spectrometer was analyzed by comparing the data against the UniProt protein data, and as a result, the antigen in question was identified as EEF1A2 binding protein-like (amino acid sequence: SEQ ID NO: 5, encoding nucleotide sequence: SEQ ID NO: 4). SEQ ID NO: 6 corresponds to amino acid 143-159 in SEQ ID NO: 5, SEQ ID NO: 7 corresponds to amino acid 395-404 in SEQ ID NO: 5, and SEQ ID NO: 8 corresponds to amino acid 427-447 in SEQ ID NO: 5.

[0191] Accordingly, in the present invention, the antigen in spot 2 can be any of (2A-a) to (2A-e) and (2B) as defined below: [0192] (2A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 5; [0193] (2A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 5; [0194] (2A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 4; [0195] (2A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 4; [0196] (2A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4; [0197] (2B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8, preferably a protein comprising at least 2, 3, or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 5-8 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0198] The proteins of (2A-a) to (2A-e) and (2B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0199] The proteins of (2A-a) to (2A-e) and (2B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 110 to 260 kDa, preferably around 120 to 160 kDa and an isoelectric point of 4.0 to 8.0, preferably an isoelectric point of 4.0 to 7.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0200] Preferably, a protein that is an antigen in spot 2 is an antigen of an allergy to fish.

[0201] (3) Antigen in Spot 3

[0202] As the result of sequence identification of the antigen in spot 3 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 11-16 were detected.

[0203] Also, the mass spectroscopic data (SEQ ID NOs: 11-17) obtained for spot 3 on a mass spectrometer was analyzed by comparing the data against the Uniprot protein data, and as a result, the antigen in question was identified as Alpha-1, 4-glucan phosphorylase (amino acid sequence: SEQ ID NO: 10, encoding nucleotide sequence: SEQ ID NO: 9). SEQ ID NO: 11 corresponds to amino acid 836-844 in SEQ ID NO: 10, SEQ ID NO: 12 corresponds to amino acid 13-29 in SEQ ID NO: 10, SEQ ID NO: 13 corresponds to amino acid 657-681 in SEQ ID NO: 10, SEQ ID NO: 14 corresponds to amino acid 471-479 in SEQ ID NO: 10, SEQ ID NO: 15 corresponds to amino acid 546-555 in SEQ ID NO: 10, and SEQ ID NO: 16 corresponds to amino acid 727-741 in SEQ ID NO: 10.

[0204] Accordingly, in the present invention, the antigen in spot 3 can be any of (3A-a) to (3A-e) and (3B) as defined below: [0205] (3A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 10; [0206] (3A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 10; [0207] (3A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 9; [0208] (3A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 9. [0209] (3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9; [0210] (3B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10-16, preferably a protein comprising at least 2, 3, 4, 5, 6 or all sequences of the amino acid sequences. More preferably, a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12, 13, 15, and 16, preferably a protein comprising at least 2, 3, 4, or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 10-16 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0211] The proteins of (3A-a) to (3A-e) and (3B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0212] The proteins of (3A-a) to (3A-e) and (3B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 80 to 160 kDa, preferably around 90 to 120 kDa and an isoelectric point of 4.0 to 10.0, preferably an isoelectric point of 5.0 to 8.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0213] Preferably, a protein that is an antigen in spot 3 is an antigen of an allergy to fish.

[0214] (4) Antigen in Spot 4

[0215] As the result of sequence identification of the antigen in spot 4 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 20-24 were detected.

[0216] Also, the mass spectroscopic data (SEQ ID NOs: 20-24) obtained for spot 4 on a mass spectrometer was analyzed by comparing the data against the Uniprot protein data, and as a result, the antigen in question was identified as Elongation factor 2 (amino acid sequence: SEQ ID NO: 19, encoding nucleotide sequence: SEQ ID NO: 18). SEQ ID NO: 20 corresponds to amino acid 831-840 in SEQ ID NO: 19, SEQ ID NO: 21 corresponds to amino acid 560-571 in SEQ ID NO: 19, SEQ ID NO: 22 corresponds to amino acid 560-572 in SEQ ID NO: 19, SEQ ID NO: 23 corresponds to amino acid 677-688 in SEQ ID NO: 19, and SEQ ID NO: 24 corresponds to amino acid 620-629 in SEQ ID NO: 19.

[0217] Accordingly, in the present invention, the antigen in spot 4 can be any of (4-a) to (4-e) and (4B) as defined below: [0218] (4A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 19; [0219] (4A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 19; [0220] (4A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 18; [0221] (4A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 18; [0222] (4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 18; [0223] (4B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24, preferably a protein comprising at least 2, 3, 4, 5, 6 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 19-24 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0224] The proteins of (4A-a) to (4A-e) and (4B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0225] The proteins of (4A-a) to (4A-e) and (4B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 80 to 160 kDa, preferably around 90 to 120 kDa and an isoelectric point of 5.0 to 11.0, preferably an isoelectric point of 6.0 to 8.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0226] Preferably, a protein that is an antigen in spot 4 is an antigen of an allergy to fish.

[0227] (5) Antigen in Spot 5

[0228] As the result of sequence identification of the antigen in spot 5 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 27-31 were detected.

[0229] Also, the mass spectroscopic data (SEQ ID NOs: 27-31) obtained for spot 5 on a mass spectrometer was analyzed by comparing the data against the Uniprot protein data, and as a result, the antigen in question was identified as Heat shock cognate 70 kDa protein (amino acid sequence: SEQ ID NO: 26, encoding nucleotide sequence: SEQ ID NO: 25). SEQ ID NO: 27 corresponds to amino acid 113-126 in SEQ ID NO: 26, SEQ ID NO: 28 corresponds to amino acid 513-526 in SEQ ID NO: 26, SEQ ID NO: 29 corresponds to amino acid 89-102 in SEQ ID NO: 26, SEQ ID NO: 30 corresponds to amino acid 584-597 in SEQ ID NO: 26, and SEQ ID NO: 31 corresponds to amino acid 138-159 in SEQ ID NO: 26.

[0230] Accordingly, in the present invention, the antigen in spot 5 can be any of (5A-a) to (5A-e) and (5B) as defined below: [0231] (5A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 26; [0232] (5A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 26; [0233] (5A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 25; [0234] (5A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 25; [0235] (5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 25; [0236] (5B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31, preferably a protein comprising at least 2, 3, 4, 5 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 26-31 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0237] The proteins of (5A-a) to (5A-e) and (5B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0238] The proteins of (5A-a) to (5A-e) and (5B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 50 to 110 kDa, preferably around 60 to 90 kDa and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 3.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0239] Preferably, a protein that is an antigen is an antigen of an allergy to fish.

[0240] (6) Antigen in Spot 6

[0241] As the result of sequence identification of the antigen in spot 6 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 34-41 were detected.

[0242] Also, the mass spectroscopic data (SEQ ID NOs: 34-41) obtained for spot 6 on a mass spectrometer was analyzed by comparing the data against the Uniprot protein data, and as a result, the antigen in question was identified as Serotransferrin (amino acid sequence: SEQ ID NO: 33, encoding nucleotide sequence: SEQ ID NO: 32). SEQ ID NO: 34 corresponds to amino acid 330-341 in SEQ ID NO: 33, SEQ ID NO: 35 corresponds to amino acid 332-341 in SEQ ID NO: 33, SEQ ID NO: 36 corresponds to amino acid 264-274 in SEQ ID NO: 33, SEQ ID NO: 37 corresponds to amino acid 113-124 in SEQ ID NO: 33, SEQ ID NO: 38 corresponds to amino acid 112-124 in SEQ ID NO: 33, SEQ ID NO: 39 corresponds to amino acid 112-125 in SEQ ID NO: 33, SEQ ID NO: 40 corresponds to amino acid 275-292 in SEQ ID NO: 33, and SEQ ID NO: 41 corresponds to amino acid 436-446 in SEQ ID NO: 33.

[0243] Accordingly, in the present invention, the antigen in spot 6 can be any of (6A-a) to (6A-e) and (6B) as defined below: [0244] (6A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 33; [0245] (6A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 33; [0246] (6A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 32; [0247] (6A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 32; [0248] (6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 32; [0249] (6B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 33-41, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 33-41 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0250] The proteins of (6A-a) to (6A-e) and (6B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0251] The proteins of (6A-a) to (6A-e) and (6B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 60 to 110 kDa, preferably around 70 to 100 kDa and an isoelectric point of 4.0 to 8.0, preferably an isoelectric point of 5.0 to 7.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0252] Preferably, a protein that is an antigen in spot 6 is an antigen of an allergy to fish.

[0253] (7) Antigen in Spot 7

[0254] As the result of sequence identification of the antigen in spot 7 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 44-54 were detected.

[0255] Also, the mass spectroscopic data (SEQ ID NOs: 44-54) obtained for spot 7 on a mass spectrometer was analyzed by comparing the data against the Uniprot protein data, and as a result, the antigen in question was identified as Myosin binding protein H-like (amino acid sequence: SEQ ID NO: 43, encoding nucleotide sequence: SEQ ID NO: 42). SEQ ID NO: 44 corresponds to amino acid 275-292 in SEQ ID NO: 43, SEQ ID NO: 45 corresponds to amino acid 49-66 in SEQ ID NO: 43, SEQ ID NO: 46 corresponds to amino acid 67-77 in SEQ ID NO: 43, SEQ ID NO: 47 corresponds to amino acid 310-323 in SEQ ID NO: 43, SEQ ID NO: 48 corresponds to amino acid 382-401 in SEQ ID NO: 43, SEQ ID NO: 49 corresponds to amino acid 402-418 in SEQ ID NO: 43, SEQ ID NO: 50 corresponds to amino acid 210-219 in SEQ ID NO: 43, SEQ ID NO: 51 corresponds to amino acid 210-228 in SEQ ID NO: 43, SEQ ID NO: 52 corresponds to amino acid 152-183 in SEQ ID NO: 43, SEQ ID NO: 53 corresponds to amino acid 100-112 in SEQ ID NO: 43, and SEQ ID NO: 54 corresponds to amino acid 113-129 in SEQ ID NO: 43.

[0256] Accordingly, in the present invention, the antigen in spot 7 can be any of (7A-a) to (7A-e) and (7B) as defined below: [0257] (7A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 43; [0258] (7A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 43; [0259] (7A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 42; [0260] (7A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 42; [0261] (7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 42; [0262] (7B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 43-54, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all sequences of the amino acid sequences; As referred to above, the amino acid sequence of any of SEQ ID NOs: 43-54 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0263] The proteins of (7A-a) to (7A-e) and (7B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0264] The proteins of (7A-a) to (7A-e) and (7B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 40 to 80 kDa, preferably around 50 to 60 kDa and an isoelectric point of 4.0 to 8.0, preferably an isoelectric point of 4.0 to 7.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0265] Preferably, a protein that is an antigen in spot 7 is an antigen of an allergy to fish.

[0266] (8) Antigen in Spot 8

[0267] As the result of sequence identification of the antigen in spot 8 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 57-59 were detected.

[0268] Also, the mass spectroscopic data (SEQ ID NOs: 57-59) obtained for spot 8 on a mass spectrometer was analyzed by comparing the data against the Uniprot protein data, and as a result, the antigen in question was identified as Desmin (Fragment) (amino acid sequence: SEQ ID NO: 56, encoding nucleotide sequence: SEQ ID NO: 55). SEQ ID NO: 57 corresponds to amino acid 364-380 in SEQ ID NO: 56, SEQ ID NO: 58 corresponds to amino acid 74-94 in SEQ ID NO: 56, and SEQ ID NO: 59 corresponds to amino acid 273-282 in SEQ ID NO: 56.

[0269] Accordingly, in the present invention, the antigen in spot 8 can be any of (8A-a) to (8A-e) and (8B) as defined below: [0270] (8A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 56; [0271] (8A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 56; [0272] (8A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 55; [0273] (8A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 55; [0274] (8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 55; [0275] (8B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59, preferably a protein comprising at least 2, 3, or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 56-59 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0276] The proteins of (8A-a) to (8A-e) and (8B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0277] The proteins of (8A-a) to (8A-e) and (8B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 40 to 80 kDa, preferably around 50 to 60 kDa and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0278] Preferably, a protein that is an antigen in spot 8 is an antigen of an allergy to fish.

[0279] (9) Antigen in Spot 9

[0280] As the result of sequence identification of the antigen in spot 9 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 62-68 were detected.

[0281] Also, the mass spectroscopic data (SEQ ID NOs: 62-68) obtained for spot 9 on a mass spectrometer was analyzed by comparing the data against the UniProt protein data, and as a result, the antigen in question was identified as Capping protein (Actin filament) muscle Z-line beta (amino acid sequence: SEQ ID NO: 61, encoding nucleotide sequence: SEQ ID NO: 60). SEQ ID NO: 62 corresponds to amino acid 200-223 in SEQ ID NO: 61, SEQ ID NO: 63 corresponds to amino acid 238-254 in SEQ ID NO: 61, SEQ ID NO: 64 corresponds to amino acid 261-274 in SEQ ID NO: 61, SEQ ID NO: 65 corresponds to amino acid 224-235 in SEQ ID NO: 61, SEQ ID NO: 66 corresponds to amino acid 169-181 in SEQ ID NO: 61, SEQ ID NO: 67 corresponds to amino acid 245-254 in SEQ ID NO: 61, and SEQ ID NO: 68 corresponds to amino acid 79-95 in SEQ ID NO: 61.

[0282] Accordingly, the antigen in spot 9 in the present invention can be any of the proteins as defined below in (9A-a) to (9A-e), and (9B): [0283] (9A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 61; [0284] (9A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 61; [0285] (9A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 60; [0286] (9A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 60; [0287] (9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 60; [0288] (9B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 61-68, preferably a protein comprising at least 2, 3, 4, 5, 6, 7 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 61-68 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids;

[0289] The proteins as defined above in (9A-a) to (9A-e), and (9B) also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0290] The proteins as defined above in (9A-a) to (9A-e), and (9B) can be proteins that are found in a protein spot with a molecular weight of around 20 to 50 kDa, preferably around 30 to 40 kDa, and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0291] Preferably, a protein that is an antigen in spot 9 is an antigen of an allergy to fish.

[0292] (10) Antigen in Spot 10

[0293] As the result of sequence identification of the antigen in spot 10 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 71-108 were detected.

[0294] Also, the mass spectroscopic data (SEQ ID NOs: 71-108) obtained for spot 10 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as myosin heavy chain, fast skeletal muscle-like (amino acid sequence: SEQ ID NO: 70, encoding nucleotide sequence: SEQ ID NO: 69). SEQ ID NO: 71 corresponds to amino acid 13-19 in SEQ ID NO:70, SEQ ID NO: 72 corresponds to amino acid 262-271 in SEQ ID NO: 70, SEQ ID NO: 73 corresponds to amino acid 996-1014 in SEQ ID NO: 70, SEQ ID NO: 74 corresponds to amino acid 951-961 in SEQ ID NO: 70, SEQ ID NO: 75 corresponds to amino acid 1293-1303 in SEQ ID NO: 70, SEQ ID NO: 76 corresponds to amino acid 1082-1091 in SEQ ID NO: 70, SEQ ID NO: 77 corresponds to amino acid 1847-1856 in SEQ ID NO:70, SEQ ID NO: 78 corresponds to amino acid 172-186 in SEQ ID NO: 70, SEQ ID NO: 79 corresponds to amino acid 919-937 in SEQ ID NO: 70, SEQ ID NO: 80 corresponds to amino acid 249-258 in SEQ ID NO: 70, SEQ ID NO: 81 corresponds to amino acid 706-717 in SEQ ID NO: 70, SEQ ID NO: 82 corresponds to amino acid 50-59 in SEQ ID NO: 70, SEQ ID NO: 83 corresponds to amino acid 415-430 in SEQ ID NO:70, SEQ ID NO: 84 corresponds to amino acid 834-845 in SEQ ID NO: 70, SEQ ID NO: 85 corresponds to amino acid 617-630 in SEQ ID NO: 70, SEQ ID NO: 86 corresponds to amino acid 1556-1567 in SEQ ID NO: 70, SEQ ID NO: 87 corresponds to amino acid 1391-1408 in SEQ ID NO: 70, SEQ ID NO: 88 corresponds to amino acid 1025-1040 in SEQ ID NO: 70, SEQ ID NO: 89 corresponds to amino acid 1813-1836 in SEQ ID NO:70, SEQ ID NO: 90 corresponds to amino acid 743-755 in SEQ ID NO: 70, SEQ ID NO: 91 corresponds to amino acid 1172-1192 in SEQ ID NO: 70, SEQ ID NO: 92 corresponds to amino acid 353-364 in SEQ ID NO: 70, SEQ ID NO: 93 corresponds to amino acid 1261-1292 in SEQ ID NO: 70, SEQ ID NO: 94 corresponds to amino acid 1783-1789 in SEQ ID NO: 70, SEQ ID NO: 95 corresponds to amino acid 1502-1519 in SEQ ID NO: 70, SEQ ID NO: 96 corresponds to amino acid 1484-1497 in SEQ ID NO:70, SEQ ID NO: 97 corresponds to amino acid 1194-1212 in SEQ ID NO: 70, SEQ ID NO: 98 corresponds to amino acid 1304-1314 in SEQ ID NO: 70, SEQ ID NO: 99 corresponds to amino acid 369-384 in SEQ ID NO: 70, SEQ ID NO: 100 corresponds to amino acid 1315-1322 in SEQ ID NO: 70, SEQ ID NO: 101 corresponds to amino acid 1536-1555 in SEQ ID NO: 70, SEQ ID NO: 102 corresponds to amino acid 237-248 in SEQ ID NO:70, SEQ ID NO: 103 corresponds to amino acid 1699-1725 in SEQ ID NO: 70, SEQ ID NO: 104 corresponds to amino acid 1092-1104 in SEQ ID NO: 70, SEQ ID NO: 105 corresponds to amino acid 407-414 in SEQ ID NO: 70, SEQ ID NO: 106 corresponds to amino acid 1897-1917 in SEQ ID NO: 70, SEQ ID NO:107 corresponds to amino acid 1458-1470 in SEQ ID NO: 70, and SEQ ID NO: 108 corresponds to amino acid 1373-1388 in SEQ ID NO: 70.

[0295] Accordingly, the antigen in spot 10 in the present invention can be any of the proteins as defined below in (10A-a) to (10A-e), and (10B): [0296] (10A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 70; [0297] (10A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 70; [0298] (10A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 69; [0299] (10A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 69; [0300] (10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 69; [0301] (10B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 70-108, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 70-108 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids;

[0302] The proteins as defined above in (10A-a) to (10A-e), and (10B) also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0303] The proteins as defined above in (10A-a) to (10A-e), and (10B) can be proteins that are found in a protein spot with a molecular weight of around 160 to 300 kDa, preferably around 160 to 260 kDa, more preferably around 200 to 230 kDa, and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0, more preferably an isoelectric point of 4.5 to 5.5 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0304] Preferably, a protein that is an antigen in spot 10 is an antigen of an allergy to fish.

[0305] (11) Antigen in Spot 11

[0306] As the result of sequence identification of the antigen in spot 11 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 111-119 were detected.

[0307] Also, the mass spectroscopic data (SEQ ID NOs: 111-119) obtained for spot 11 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as glycogen phosphorylase, muscle form-like (amino acid sequence: SEQ ID NO: 110, encoding nucleotide sequence: SEQ ID NO: 109). SEQ ID NO: 111 corresponds to amino acid 471-479 in SEQ ID NO: 110, SEQ ID NO: 112 corresponds to amino acid 547-555 in SEQ ID NO: 110, SEQ ID NO: 113 corresponds to amino acid 203-215 in SEQ ID NO:110, SEQ ID NO: 114 corresponds to amino acid 727-741 in SEQ ID NO: 110, SEQ ID NO: 115 corresponds to amino acid 508-520 in SEQ ID NO: 110, SEQ ID NO: 116 corresponds to amino acid 742-755 in SEQ ID NO: 110, SEQ ID NO: 117 corresponds to amino acid 13-29 in SEQ ID NO: 110, SEQ ID NO:118 corresponds to amino acid 775-784 in SEQ ID NO: 110, and SEQ ID NO: 119 corresponds to amino acid 643-656 in SEQ ID NO: 110.

[0308] Accordingly, the antigens in spot 11 in the present invention can be any of the proteins as defined below in (11A-a) to (11A-e) and (11B): [0309] (11A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 110; [0310] (11A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 110; [0311] (11A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 109; [0312] (11A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 109; [0313] (11A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 109; [0314] (11B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 110-119, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 110-119 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0315] The proteins of (11A-a) to (11A-e) and (11B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0316] The proteins of (11A-a) to (11A-e) and (11B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 80 to 160 kDa, preferably around 80 to 110 kDa, more preferably around 90 to 110 kDa and an isoelectric point of 4.0 to 8.0, preferably an isoelectric point of 5.0 to 7.5, more preferably an isoelectric point of 6.5 to 7.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0317] Preferably, a protein that is an antigen in spot 11 is an antigen of an allergy to fish.

[0318] (12) Antigen in Spot 12

[0319] As the result of sequence identification of the antigen in spot 12 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 122-136 were detected.

[0320] Also, the mass spectroscopic data (SEQ ID NOs: 122-136) obtained for spot 12 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as myosin-binding protein C, fast-type-like (amino acid sequence: SEQ ID NO: 121, encoding nucleotide sequence: SEQ ID NO: 120). SEQ ID NO: 122 corresponds to amino acid 197-204 in SEQ ID NO: 121, SEQ ID NO: 123 corresponds to amino acid 421-434 in SEQ ID NO: 121, SEQ ID NO: 124 corresponds to amino acid 329-336 in SEQ ID NO:121, SEQ ID NO: 125 corresponds to amino acid 413-420 in SEQ ID NO: 121, SEQ ID NO:126 corresponds to amino acid 240-246 in SEQ ID NO: 121, SEQ ID NO: 127 corresponds to amino acid 214-228 in SEQ ID NO: 121, SEQ ID NO: 128 corresponds to amino acid 1014-1024 in SEQ ID NO: 121, SEQ ID NO: 129 corresponds to amino acid 842-855 in SEQ ID NO: 121, SEQ ID NO: 130 corresponds to amino acid 260-274 in SEQ ID NO:121, SEQ ID NO: 131 corresponds to amino acid 672-685 in SEQ ID NO: 121, SEQ ID NO: 132 corresponds to amino acid 506-512 in SEQ ID NO: 121, SEQ ID NO: 133 corresponds to amino acid 205-213 in SEQ ID NO: 121, SEQ ID NO: 134 corresponds to amino acid 165-182 in SEQ ID NO: 121, SEQ ID NO:135 corresponds to amino acid 321-328 in SEQ ID NO: 121, and SEQ ID NO: 136 corresponds to amino acid 70-82 in SEQ ID NO: 121.

[0321] Accordingly, the antigens in spot 12 in the present invention can be any of the proteins as defined below in (12A-a) to (12A-e) and (12B): [0322] (12A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 121; [0323] (12A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 121; [0324] (12A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 119; [0325] (12A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 119; [0326] (12A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 119; [0327] (12B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 121-136, preferably a protein comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 121-136 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0328] The proteins of (12A-a) to (12A-e) and (12B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0329] The proteins of (12A-a) to (12A-e) and (12B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 100 to 160 kDa, preferably around 110 to 150 kDa, more preferably around 120 to 140 kDa and an isoelectric point of 4.0 to 7.0, preferably an isoelectric point of 4.0 to 6.0, more preferably an isoelectric point of 5.0 to 6.0 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0330] Preferably, a protein that is an antigen in spot 12 is an antigen of an allergy to fish.

[0331] (13) Antigen in Spot 13

[0332] As the result of sequence identification of the antigen in spot 13 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 139-142 were detected.

[0333] Also, the mass spectroscopic data (SEQ ID NOs: 139-142) obtained for spot 13 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as ATP synthase subunit beta, mitochondrial (amino acid sequence: SEQ ID NO: 138, encoding nucleotide sequence: SEQ ID NO: 137). SEQ ID NO: 139 corresponds to amino acid 191-201 in SEQ ID NO: 138, SEQ ID NO: 140 corresponds to amino acid 449-469 in SEQ ID NO: 138, SEQ ID NO:141 corresponds to amino acid 202-214 in SEQ ID NO: 138, and SEQ ID NO: 142 corresponds to amino acid 178-187 in SEQ ID NO: 138.

[0334] Accordingly, the antigen in spot 13 in the present invention can be any of the proteins as defined below in (13A-a) to (13A-e), and (13B): [0335] (13A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 138; [0336] (13A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 138; [0337] (13A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 137; [0338] (13A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 137; [0339] (13A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 137; [0340] (13B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 138-142, preferably a protein comprising at least 2, 3, 4 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 138-142 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids;

[0341] The proteins as defined above in (13A-a) to (13A-e), and (13B) also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0342] The proteins as defined above in (13A-a) to (13A-e), and (13B) can be proteins that are found in a protein spot with a molecular weight of around 30 to 70 kDa, preferably around 40 to 60 kDa, more preferably around 45 to 55 kDa and an isoelectric point of 3.0 to 7.0, preferably an isoelectric point of 3.0 to 6.0, more preferably an isoelectric point of 4.0 to 5.5 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0343] Preferably, a protein that is an antigen in spot 13 is an antigen of an allergy to fish.

[0344] (14) Antigen in Spot 14

[0345] As the result of sequence identification of the antigen in spot 14 by mass spectroscopy, the following amino acid sequences of SEQ ID NOs: 145-149 were detected.

[0346] Also, the mass spectroscopic data (SEQ ID NOs: 145-149) obtained for spot 14 on a mass spectrometer was analyzed by comparing the data against the NCBI protein data, and as a result, the antigen in question was identified as L-lactate dehydrogenase A chain-like (amino acid sequence: SEQ ID NO: 144, encoding nucleotide sequence: SEQ ID NO: 143). SEQ ID NO: 145 corresponds to amino acid 119-126 in SEQ ID NO: 144, SEQ ID NO: 146 corresponds to amino acid 7-22 in SEQ ID NO: 144, SEQ ID NO: 147 corresponds to amino acid 9-22 in SEQ ID NO: 144, SEQ ID NO: 148 corresponds to amino acid 270-278 in SEQ ID NO: 144, and SEQ ID NO: 149 corresponds to amino acid 91-118 in SEQ ID NO: 144.

[0347] Accordingly, the antigens in spot 14 in the present invention can be any of the proteins as defined below in (14A-a) to (14A-e) and (14B): [0348] (14A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 144; [0349] (14A-b) a protein comprising an amino acid sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 144; [0350] (14A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID. NO: 143; [0351] (14A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence of SEQ ID NO: 143; [0352] (14A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 143; [0353] (14B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144-149, preferably a protein comprising at least 2, 3, 4, 5 or all sequences of the amino acid sequences. As referred to above, the amino acid sequence of any of SEQ ID NOs: 144-149 may be an amino acid sequence derived therefrom by deletion, substitution, insertion or addition of one or several amino acids.

[0354] The proteins of (14A-a) to (14A-e) and (14B) as defined above also include those proteins whose amino acid residues are modified by phosphorylation, sugar chain modification, aminoacylation, ring-opening, deamination or the like.

[0355] The proteins of (14A-a) to (14A-e) and (14B) as defined above can be proteins that are found in a protein spot with a molecular weight of around 20 to 50 kDa, preferably around 30 to 50 kDa, more preferably around 30 to 40 kDa and an isoelectric point of 5.0 to 9.0, preferably an isoelectric point of 6.0 to 8.0, more preferably an isoelectric point of 6.5 to 7.5 on gels used in the two-dimensional electrophoresis performed under the conditions described above in the subsection titled "Identification of antigens".

[0356] Preferably, a protein that is an antigen in spot 14 is an antigen of an allergy to fish.

[0357] By stating herein "deletion, substitution, insertion or addition of one or several amino acids" in relation to amino acid sequence, it is meant that in an amino acid sequence of interest, one or several amino acids (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2%, or 1% of amino acids with respect to the total length of the amino acid sequence) are deleted, one or several amino acids are substituted by any other amino acids, any other amino acids are inserted, and/or any other amino acids are added.

[0358] Among the aforementioned modifications, substitution is preferably conservative substitution. The "conservative substitution" refers to the substitution of a certain amino acid residue by a different amino acid residue having similar physicochemical characteristics, and can be any type of substitution as long as it does not substantially change the characteristics of the structure of the original sequence for example, any type of substitution is acceptable as long as any substituted amino acids do not disrupt the helical structure of the original sequence or other secondary structures that characterize the original sequence. The following gives examples of separate groups of amino acid residues that are conservatively substitutable with each other, but substitutable amino acid residues are not limited to the examples given below. [0359] Group A: leucine, isoleucine, valine, alanine, methionine [0360] Group B: aspartic acid, glutamic acid [0361] Group C: asparagine, glutamine [0362] Group D: lysine, arginine [0363] Group E: serine, threonine [0364] Group F: phenylalanine, tyrosine

[0365] In the case of non-conservative substitution, one member belonging to one of the aforementioned groups can be replaced with a member belong to any other group. For example, in order to eliminate the possibility of unwanted sugar-chain modification, amino acids of group B, D or E as listed above may be substituted by those of any other group. Also, cysteines may be deleted or substituted by any other amino acids to prevent them from being folded into a protein in its tertiary structure. Also, in order to maintain the balance between hydrophilicity and hydrophobicity or to increase hydrophilicity for the purpose of facilitating synthesis, any amino acids may be substituted in consideration of the hydropathy scales of amino acids, which are a measure of the hydrophilic and hydrophobic properties of amino acids (J. Kyte and R. Doolittle, J. Mol. Biol., Vol.157, p.105-132, 1982).

[0366] As referred to herein, the percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity can be determined using a computer program. Examples of such computer programs include BLAST and ClustalW. In particular, various conditions (parameters) for identity searches with the BLAST program are described in Altschul, et al. (Nucl. Acids. Res., 25, p.3389-3402, 1997), and are publicly available on the websites of the National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDBJ) (Altschul, et al., BLAST Manual, Altschul, et al., NCB/NLM/NIH Bethesda, Md. 20894). Also, the percent identity can be determined using a genetic information processing software program, such as GENETYX Ver.7 (Genetyx Corporation), DINASIS Pro (Hitachi Software Engineering Co., Ltd.), or Vector NTI (Infomax Inc.).

[0367] By stating herein "deletion, substitution, insertion or addition of one or several nucleotides" in relation to nucleotide sequence, it is meant that in a nucleotide sequence of interest, one or several nucleotides (e.g., 30%, preferably 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1%, of nucleotides with respect to the total length of the nucleotide sequence) are deleted, one or several nucleotides are substituted by any other nucleotides, any other nucleotides are inserted, and/or any other nucleotides are added. It is preferable that such a nucleotide deletion, substitution, insertion or addition should not give rise to a frame shift in an amino acid coding sequence.

[0368] As referred to herein, the percent identity between two nucleotide sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity can be determined using a computer program. Examples of such sequence comparison computer programs include the BLASTN program, version 2.2.7, available on the website of the National Library of Medicine (http://www.ncbi.nlm.nih.gov/blast/b12seq/b1s.html) (Altschul, et al. (1990) J. Mol. Biol., 215: 403-10), or the WU-BLAST 2.0 algorithm. Standard default parameter settings for WU-BLAST 2.0 are found and available on the following website: http://blast.wustl.edu.

[0369] As referred to herein, "under stringent conditions" means that hybridization takes place under moderately or highly stringent conditions. To be specific, the moderately stringent conditions can be easily determined by those having ordinary skill in the art on the basis of, for example, the length of DNA. Basic conditions are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd ed., ch.6-7, Cold Spring Harbor Laboratory Press, 2001. The moderately stringent conditions include hybridization under the conditions of preferably 1.times.SSC to 6.times.SSC at 42.degree. C. to 55.degree. C., more preferably 1.times.SSC to 3.times.SSC at 45.degree. C. to 50.degree. C., most preferably 2.times.SSC at 50.degree. C. In the case of using a hybridization solution containing, for example, about 50% formamide, a temperature around 5 to 15.degree. C. lower than the foregoing should be adopted. Washing is also carried out under the conditions of 0.5.times.SSC to 6.times.SSC at 40.degree. C. to 60.degree. C. In the process of hybridization and washing, generally 0.05% to 0.2% SDS, preferably about 0.1% SDS, may be added. Likewise, the highly stringent conditions can be easily determined by those having ordinary skill in the art on the basis of, for example, the length of DNA. Generally, the highly stringent (high stringent) conditions include hybridization and/or washing at a higher temperature and/or a lower salt concentration than those adopted under the moderately stringent conditions. For example, hybridization is carried out under the conditions of 0.1.times.SSC to 2.times.SSC at 55.degree. C. to 65.degree. C., more preferably 0.1.times.SSC to 1.times.SSC at 60.degree. C. to 65.degree. C., most preferably 0.2.times.SSC at 63.degree. C. Washing is carried out under the conditions of 0.2.times.SSC to 2.times.SSC at 50.degree. C. to 68.degree. C., more preferably 0.2.times.SSC at 60 to 65.degree. C.

[0370] Antigens may be obtained by separating and purifying them from fish using a combination of protein purification methods well known to those skilled in the art. Also, antigens may be obtained by expressing them as recombinant proteins using a genetic recombination technique well known to those skilled in the art and by separating and purifying them using protein purification methods well known to those skilled in the art.

[0371] Exemplary protein purification methods include: solubility-based purification methods such as salt precipitation and solvent precipitation; purification methods based on molecular weight difference, such as dialysis, ultrafiltration, gel filtration and SDS-PAGE; charge-based purification methods such as ion exchange chromatography and hydroxylapatite chromatography; specific affinity-based purification methods such as affinity chromatography; purification methods based on hydrophobicity difference, such as reverse-phase high-performance liquid chromatography; and purification methods based on isoelectric point difference, such as isoelectric focusing.

[0372] Preparation of a protein by a genetic recombination technique is carried out by preparing an expression vector comprising an antigen-encoding nucleic acid, introducing the expression vector into appropriate host cells by gene transfer or genetic transformation, culturing the host cells under suitable conditions for expression of a recombinant protein, and recovering the recombinant protein expressed in the host cells.

[0373] The "vector" refers to a nucleic acid that can be used to introduce a nucleic acid attached thereto into host cells. The "expression vector" is a vector that can induce the expression of a protein encoded by a nucleic acid introduced thereby. Exemplary vectors include plasmid vectors, viral vectors, and the like. Those skilled in the art can select an appropriate expression vector for the expression of a recombinant protein depending on the type of host cells to be used. To facilitate purification, an affinity tags such as 6 His residues may be included. Moreover, a protein may be synthesized so that a signal sequence is included and the protein is secreted out of cells.

[0374] The "host cells" refers to cells that undergo gene transfer or genetic transformation by a vector. The host cells can be appropriately selected by those skilled in the art depending on the type of the vector to be used. The host cells can be derived from prokaryotes such as Escherichia coli (E. coli). When prokaryotic cells like E. coli are used as host cells, the antigen of the present invention may be designed to contain an N-terminal methionine residue in order to facilitate the expression of a recombinant protein in the prokaryotic cells. The N-terminal methionine can be cleaved from the recombinant protein after expression. Also, the host cells may be cells derived from eukaryotes, such as single-cell eukaryotes like yeast, plant cells and animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, murine cells or insect cells) or silkworm.

[0375] Gene transfer or genetic transformation of an expression vector into host cells can be carried out as appropriate by following a technique known to those skilled in the art. Those skilled in the art can make possible the expression of a recombinant protein by selecting suitable conditions for the expression of the recombinant protein as appropriate depending on the type of host cells and culturing the host cells under the selected conditions. Then, those skilled in the art can homogenize the host cells having the expressed recombinant protein, and separate and purify an antigen expressed as the recombinant protein from the resulting homogenate by using an appropriate combination of such protein purification methods as mentioned above.

[0376] Diagnostic Kit and Method

[0377] The present invention provides a method for providing an indicator for diagnosing a fish allergy in a subject, the method comprising the steps of: [0378] (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody; [0379] (ii) detecting binding between an IgE antibody present in the sample obtained from the subject and the antigen; and [0380] (iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject has a fish allergy is provided; [0381] wherein the antigen is at least one of the proteins identified as the aforementioned antigens (1) to (9) or at least one of the proteins identified as the aforementioned antigens (10) to (14).

[0382] The sample obtained from a subject is a solution containing an IgE antibody, as collected from the subject. Examples of such solutions include blood, saliva, sputum, snivel, urine, sweat, and tear. The sample obtained from the subject may be subjected to pretreatment for increasing the concentration of an IgE antibody in the sample before being contacted with an antigen. The pretreatment of a sample may involve, for example, collection of the serum and the plasma from the blood. Furthermore, Fab portions that are binding portions with the antigens may be purified. In a particularly preferred embodiment, the step (i) mentioned above is carried out by contacting an IgE antibody present in the serum obtained from a subject with an antigen.

[0383] The IgE antibody may be the IgE antibody itself or mast cells or the like to which the IgE antibody is bound.

[0384] Detection of contact and binding between the sample obtained from a subject and an antigen can be carried out by using a known method. Examples of such methods that can be used include detection by ELISA (Enzyme-Linked Immunosorvent Assay), sandwich immunoassay, immunoblotting, immunoprecipitation, or immunochromatography. These are all techniques involving contacting and binding an IgE antibody from a subject with an antigen and detecting the IgE antibody specifically bound to the antigen. Examples thereof include methods involving bringing an enzyme-labeled secondary antibody in contact, adding a substrate (usually a chromogenic or luminescent reagent) of the enzyme, and detecting the product of the enzymatic reaction; methods involving bringing a biotinylated secondary antibody in contact, adding an avidin-conjugated dye, and detecting the dye; and methods involving detecting a fluorescently labeled secondary antibody. Also, detection by a measurement method that permits the evaluation of binding between an antigen and an IgE antibody, such as surface plasmon resonance (SPR), can be used. A plurality of antigen-specific IgE antibodies may be mixed.

[0385] The antigen may be provided as an isolated antigen in a state immobilized on a carrier. In this case, the steps (i) and (ii) mentioned above can be carried out using ELISA, sandwich immunoassay, immunochromatography, surface plasmon resonance, or the like. Also, the step (i) mentioned above is carried out by contacting the sample obtained from a subject with an antigen-immobilized surface. The isolated antigen may be obtained by separating and purifying it from fish using a combination of protein purification methods well known to those skilled in the art, or by preparing it using a genetic recombination technique. Moreover, the antibody that binds to the antigen may be in an immobilized state.

[0386] The antigen may be in an unimmobilized state to a carrier. In this case, the steps (1) and (ii) mentioned above can be carried out by flow cytometry or the like and the presence of the antigen bound to an antibody may be confirmed using a laser beam. Examples include basophil activation test (BAT) in which an antigen binds to an antibody and the amount of the surface antigen CD203c which is expressed when basophils in the sample are activated is measured, and the like. Another example is histamine release test (HRT) in which it is examined whether histamine is released or not by bringing an antigen in contact with hemocytes via the binding to an antibody in a sample.

[0387] The antigen may be transferred from a state separated by two-dimensional electrophoresis and detected by immunoblotting. The two-dimensional electrophoresis is a technique for separating a protein sample by performing isoelectric focusing in the first dimension and performing SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in the second dimension. The conditions for two-dimensional electrophoresis are not particularly limited as long as the conditions permit the separation of the antigen of the present invention. For example, the conditions for two-dimensional electrophoresis as described above in the subsection titled "Identification of antigens" can be adopted. Also, the electrophoresis conditions may be determined by reference to the descriptions in PTLs 1 to 4 mentioned above. For example, two-dimensional electrophoresis can be carried out under the conditions that satisfy at least one selected from the group consisting of the following requirements: [0388] (A) the isoelectric focusing gels used in the first dimension should have a gel-strip length of 5 to 10 cm and a gel pH range of 3 to 10, and the pH gradient of the gels in the direction of electrophoresis should be as follows: where the gel-strip length up to pH 5 is taken as "a", that length from pH 5 to 7 as "b", and that length above pH 7 as "c", the relations "a<b" and "b>c" are satisfied; [0389] (B) in the case of (A), when the total gel-strip length is taken as 1, "a" should be in the range of 0.15 to 0.3, "b" should be in the range of 0.4 to 0.7, and "c" should be in the range of 0.15 to 0.3; [0390] (C) in the first dimensional isoelectric focusing, a constant voltage step should be performed by applying a constant voltage ranging from 100 V to 600 V per gel strip containing a sample, and after the electrophoresis variation width during electrophoresis for 30 minutes falls within the range of 5 .mu.A, a voltage-increasing step should be started at which the voltage is increased from the aforementioned constant voltage; [0391] (D) in the case of (C), the final voltage at the voltage-increasing step should be in the range of 3000 V to 6000 V; [0392] (E) the isoelectric focusing gels used in the first dimension should have a longitudinal gel-strip length of 5 to 10 cm, and the electrophoresis gels used in the second dimension should have a gel concentration at the distal end in the direction of electrophoresis, which is in the range of 3 to 6%; and [0393] (F) in the case of (E), the electrophoresis gels used in the second dimension should have a gel concentration at the proximal end in the direction of electrophoresis, which is set to a higher value than that at the distal end in the direction of electrophoresis.

[0394] The aforementioned antigens (1) to (9) and the aforementioned antigens (10) to (14) are antigens which specifically bind to IgE antibodies in patients with an allergy to fish. Therefore, when binding between an IgE antibody from a subject and the antigen is detected, an indicator of the fact that the subject has a fish allergy is provided.

[0395] The present invention further provides a kit for diagnosing an allergy to a fish, comprising at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14). The diagnostic kit of this invention may be used in the aforementioned method for providing an indicator for diagnosing an allergy to a fish or in a diagnostic method as described later. The diagnostic kit of this invention comprises at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) and may comprise a chromogenic substrate or luminescent substrate serving as a substrate of an enzyme-labeled anti-IgE antibody and the enzyme or a biotinylated anti-IgE antibody and an avidin-conjugated dye that binds to the biotin. Also, a fluorescent-labeled anti-IgE antibody may be used. In the diagnostic kit of this invention, the antigen may be provided in a state immobilized on a carrier. The diagnostic kit of this invention may also be provided together with instructions on the procedure for diagnosis or a package containing said instructions.

[0396] In another embodiment, the diagnostic kit comprises a companion diagnostic agent for an allergy to fish. The companion diagnostic agent is used to analyze the reactivity of a pharmaceutical product for the purpose of identifying a patient in which the pharmaceutical product is expected to work or a patient at risk for serious side effects from the pharmaceutical product, or optimizing a therapy using the pharmaceutical product. Here, examples of optimizing a therapy include determination of the dosage and administration, decision of stopping the administration, determination of the allergen component to be used for the induction of immune tolerance, and the like.

[0397] The present invention further provides a composition for diagnosing an allergy to a fish, comprising at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14). The diagnostic composition of this invention can be used in a diagnostic method as described below. The diagnostic composition of this invention may further comprise a pharmaceutically acceptable carrier and/or additives commonly used with the antigen of this invention depending on the need.

[0398] In one embodiment, the present invention provides a method for diagnosing an allergy to a fish in a subject, the method comprising: [0399] (i) contacting a sample obtained from the subject with an antigen; [0400] (ii) detecting binding between an IgE antibody present in the sample obtained from the subject and the antigen; and [0401] (iii) when the binding between the IgE antibody in the subject and the antigen is detected, diagnosing the subject as being allergic to a fish; [0402] wherein the antigen is at least one of the proteins identified as the aforementioned antigens (1) to (9) or at least one of the proteins identified as the aforementioned antigens (10) to (14). In this method, each of the steps (i) and (ii) is performed as described above regarding each of the corresponding steps of the method for providing an indicator for diagnosing an allergy to a fish.

[0403] In another embodiment, the present invention provides a method for diagnosing an allergy to a fish in a subject, the method comprising administering to the subject at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14). This method may be performed in the form of a skin test characterized by applying the antigen onto the skin. Examples of the skin test include various forms of tests, such as: a prick test in which a diagnostic composition is applied onto the skin and then a tiny prick to such an extent as not to provoke bleeding is made in the skin to allow an antigen to infiltrate into the skin, thereby observing a skin reaction; a scratch test in which a diagnostic composition is applied onto the skin and then the skin is lightly scratched to observe a reaction; a patch test in which a diagnostic composition in the form of cream, ointment, etc. is applied onto the skin to observe a reaction; and an intracutaneous test in which an antigen is administered intracutaneously to observe a reaction. The method for infiltrating a diagnostic composition into the skin in a prick test or a scratch test may be a method involving bringing a tip of a lancet in contact with a diagnostic composition and wounding the skin by pricking the skin with the contact site to infiltrate the composition. If a skin reaction such as swelling occurs in a skin portion to which the antigen has been applied, the subject of interest is diagnosed as having an allergy to a fish. The amount of the antigen to be applied to the skin in such tests can be, for example, not more than 100 .mu.g per dose.

[0404] In the allergic diagnosis, a load test for the purpose of identifying the antigen is often performed. At least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) may be used as an active ingredient of a load test for diagnosing an allergy to fish. Here, the antigen protein to be used in the load test may be a protein that has been expressed and purified and may be a protein that has been expressed in a food and a food material, for example, rice-based vaccine produced by transforming rice with a gene of a cedar pollen antigen to express the antigen protein therein.

[0405] In yet another embodiment, the present invention provides at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) for use in the diagnosis of an allergy to a fish. Here, the present invention encompasses the provision of at least one of the aforementioned antigens (1) to (9) or (10) to (14) mixed with a known antigen.

[0406] In still another embodiment, the present invention provides a use of at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) for the production of a diagnostic agent for an allergy to a fish.

[0407] Pharmaceutical Composition and Treatment Method

[0408] The present invention provides a pharmaceutical composition comprising at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14).

[0409] In one embodiment, the aforementioned pharmaceutical composition is used for the treatment of an allergy to fish.

[0410] The present invention also provides a method for treating an allergy to fish, the method comprising administering at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) to a patient in need of a treatment for an allergy to a fish.

[0411] In another embodiment, the present invention provides at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) for use in the treatment of an allergy to fish. In yet another embodiment, the present invention provides use of at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) for the production of a therapeutic agent for an allergy to fish.

[0412] In the process of allergy treatment, a hyposensitization therapy aiming at inducing immunological tolerance by administering an antigen to a patient is often adopted. At least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) can be used as an active ingredient for a hyposensitization therapy for allergy to fish. Here, the antigen protein to be used in the hyposensitization therapy may be a protein that has been expressed and purified and may be a protein that has been expressed in a food and a food material, for example, rice-based vaccine produced by transforming rice with a gene of a cedar pollen antigen to express the antigen protein therein.

[0413] The pharmaceutical composition of the present invention can be administered by common administration routes. Examples of common administration routes include oral, sublingual, percutaneous, intracutaneous, subcutaneous, intravascular, intranasal, intramuscular, intraperitoneal, and intrarectal administrations.

[0414] The pharmaceutical composition of the present invention can be used as a pharmaceutical composition to which a commonly used pharmaceutically acceptable adjuvant or excipient or any other additives (e.g., stabilizer, solubilizer, emulsifier, buffer, preservative, colorant) are added by a conventional method together with the antigen of this invention depending on the need. The dosage form of the pharmaceutical composition can be selected by those skilled in the art as appropriate depending on the administration route. The pharmaceutical composition can be in the form of, for example, tablet, capsule, troche, sublingual tablet, parenteral injection, intranasal spray, poultice, solution, cream, lotion, or suppository. The administration dose, frequency and/or period of the pharmaceutical composition of this invention can be selected by a physician as appropriate depending on the administration route and the patient's condition and characteristics such as age and body weight. For example, the pharmaceutical composition may be administered to an adult patient at a dose of not more than 100 .mu.g per dose. The administration interval can be, for example, once daily, once weekly, twice monthly, once per three months or so. The administration period can be, for example, several weeks to several years. The pharmaceutical composition may be administered in such a manner that the dose is increased in incremental steps over the administration period.

[0415] Tester

[0416] The present invention provides a tester comprising an antibody for at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14).

[0417] The antibody can be prepared by a conventional method: For example, the antibody may be prepared by immunizing a mammal such as rabbit with one of the aforementioned antigens (1) to (9) or one of the aforementioned antigens (10) to (14). The antibody may be an IgE antibody, a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment thereof (e.g., Fab, F(ab').sub.2, Fab').

[0418] Further, in the aforementioned tester, the antibody may be provided in a form bound to a carrier. The type of the carrier is not particularly limited as long as it is usable for detection of binding between an antibody and an antigen. Any given carrier known to those skilled in the art can be used.

[0419] Examples of a method for determining the presence or absence of an antigen include: [0420] A method in which a prepared tester comprising an IgE antibody is contacted with a sample obtained from a food or food material, and ELISA or the like method is, for example, used to detect whether there is a binding between the IgE antibody and an antigen in the sample, and if the binding between the IgE antibody and the antigen is detected, it is determined that the antibody remains in the food or food material, of interest. [0421] A method in which proteins are extracted from a food or food material, and the proteins are subjected to electrophoresis, and it is detected with an antibody whether there is a spot band of the antigen. [0422] A method in which a food or food material is infiltrated into a filter paper or the like and an antibody solution is reacted with the filter paper to detect an antigen present in the food.

[0423] The present invention encompasses, in another embodiment, a tester for determining the presence or absence of an antigen for an allergy to fish in an object of interest, the tester comprising a primer having a nucleotide sequence complementary to at least one part of one of the nucleotide sequences set forth in SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143. Without limiting, the primer has a nucleotide sequence complementary to preferably 12 residues, 15 nucleotides, 20 nucleotides, or 25 nucleotides in the sequence of a 3' terminal or middle portion of at least one part of one sequence of the nucleotide sequences set forth in, for example, SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143. Particularly when mRNA is targeted, the tester comprises a poly A tail-complementarity primer. In a preferred embodiment, the tester comprising the primer may further comprise a primer comprising a nucleotide sequence of a 5' terminal portion of at least one sequence of the nucleotide sequences set forth in SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143, preferably a nucleotide sequence consisting of 12 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides.

[0424] For example, the presence or absence of the antigen is determined by amplifying DNA or cDNA by polymerase chain reaction (PCR) including RT-PCR by using the aforementioned primer with DNA or mRNA obtained from fish as a template and comparing the amplified DNA or cDNA sequence(s) with SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143. Examples of the method of the amplification of mRNA by PCR include RACE and the like. In the determination, the antigen is determined to be present when the comparison between the amplified DNA or cDNA and SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143 indicates the presence of point mutation(s) encoding the same amino acid(s), or when the amino acid sequence encoded by the amplified DNA or cDNA has 70% or more, preferably 80, 90, 95, 98, or 99% or more identity with an amino acid sequence of SEQ ID NO: 2, 5, 10, 19, 26, 33, 43, 56, 61, 70, 110, 121, 138, or 144, even if the nucleotide sequence of the amplified DNA or cDNA is modified from any of the nucleotide sequences of SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143 by insertion, deletion, substitution, or addition of nucleotide(s).

[0425] In one embodiment, the aforementioned tester is used to determine the presence or absence of an antigen in food materials (fish or fish eggs) or in products of interest in a food production line. The tester may also be used for quality inspection of production lines and pre-shipment products by manufacturers, or may be used for self-checking of the presence or absence of an antigen in a food or food material of interest by consumers.

[0426] Allergen-Free Food and the Like

[0427] The present invention provides fish or fish eggs in which at least one of the aforementioned antigens (1) to (9) or at least one of the aforementioned antigens (10) to (14) is eliminated or reduced, a processed product of the fish or the fish eggs or fish which lay the fish eggs or have hatched from the eggs.

[0428] The method for eliminating or reducing the antigen of the present invention in fish, fish eggs, the processed product of the fish or the fish eggs or the fish which lay the fish eggs or have hatched from the eggs is not limited. The elimination or reduction of the antigen of the present invention may be conducted by any method, as long as the method permits the elimination or reduction of the antigen.

[0429] For example, the fish or fish eggs in which the antigen of the present invention is eliminated or reduced may be obtained by preparing fish or fish eggs in which the expression of the antigen of the present invention is knocked out, using a gene knock-out technique.

[0430] As the gene knock-out technique, there can be used any methods known to those skilled in the art. For example, Oishi, et al. (Scientific Reports, Vol.6, Article number: 23980, 2016, doi:10.1038/srep23980) describes that the genome editing technique CRISPER/Cas9 is applied to primordial germ cells to obtain individual animals deficient in an allergen gene. The fish or fish eggs devoid of the antigen of this invention may also be obtained by using the same technique. Moreover, fish or fish eggs in which the antigen of the present invention is eliminated or reduced may be obtained by mating by artificial insemination with fish or fish eggs that do not contain the antigen or contains a low content of the antigen. The artificial mating of fish or fish eggs can be performed by a conventional method.

[0431] The processed product of the fish or fish eggs in which the antigen of the present invention is eliminated or reduced may be a processed product made from fish in which the antigen of the present invention is eliminated or reduced. In the case of using ordinary fish or fish eggs as a source ingredient, a treatment for removing or reducing the antigen of the present invention is performed before or after preparation of a processed product of fish or fish eggs. Examples of a method of eliminating or reducing the antigen of the present invention in a processed product of fish or fish eggs made from ordinary fish or fish eggs include methods for eliminating the protein component in a food or a food material, such as elution with a high pressure treatment and a neutral salt solution and hot steam and methods for hydrolysis, denaturation, or amino acid modification (chemical modification or elimination of side chains) by heat treatment and acid treatment. The fish in which the antigen of the present invention is eliminated or reduced may be fish that have hatched and grown from the aforementioned fish eggs in which the antigen of the present invention is eliminated or reduced. Moreover, the fish eggs in which the antigen of the present invention is eliminated or reduced may be those obtained from the aforementioned fish in which the antigen of the present invention is eliminated or reduced.

EXAMPLES

[0432] The following describes examples of the present invention. The technical scope of this invention is not limited by these examples.

Example 1: Confirmation of a Protein Pattern

[0433] Proteins contained in salmon were investigated using a two-dimensional electrophoresis method described below.

[0434] Protein Extraction

[0435] The extraction and the purification of proteins contained in salmon were performed as follows. A solubilization agent (Mammalian Lysis Buffer (MCLI), SIGMA) was added to flesh of salmon and proteins were extracted, and then a urea buffer was added to obtain a protein extract. The composition of the urea buffer is as follows.

[0436] 30 mM Tris

[0437] 2 M Thiourea

[0438] 7 M Urea

[0439] 4% (w/v) CHAPS:

[0440] 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate

[0441] Moderate amount of dilute hydrochloric acid

[0442] Distilled water was added and the total volume was adjusted to 100 mL. pH was 8.5.

[0443] Subsequently, 25 .mu.g each in terms of protein weight was mixed to obtain an extract.

[0444] Thereafter, the precipitation procedure was repeated twice using a 2D-CleanUP Kit (produced by GE). In the first round of precipitation, the collected liquid protein extract was precipitated by adding TCA (trichloroacetic acid) thereto and the precipitated product produced by this procedure (TCA-precipitated product) was collected. In the second round of precipitation, the TCA-precipitated product collected above was further precipitated by adding acetone thereto and the precipitated product (sample) produced by this procedure was collected.

[0445] Preparation of a Sample Solution

[0446] Part of the collected sample (40 .mu.g on a protein weight basis) was dissolved in 150 .mu.L of a DeStreak Rehydration Solution (produced by GE), which is a swelling buffer for first-dimensional isoelectric focusing gels, thereby obtaining a sample solution for first-dimensional isoelectric focusing (sample solution for swelling). The constituents of the DeStreak Rehydration Solution are as mentioned below.

[0447] 7M thiourea

[0448] 2M urea

[0449] 4% (w/v) of CHAPS

[0450] 0.5% (v/v) IPG buffer; produced by GE

[0451] Moderate amount of BPB (bromophenol blue)

[0452] Impregnation of the Sample into First-Dimensional Isoelectric Focusing Gels

[0453] First-dimensional isoelectric focusing gel strips (Immobiline Drystrip IPG gels (pH 3-10NL); produced by GE) were immersed in 140 .mu.L of the foregoing sample solution for first-dimensional isoelectric focusing (sample solution for swelling) and impregnated with the solution at room temperature overnight.

[0454] In this example, an IPGphor electrophoresis system produced by GE was used.

[0455] An electrophoresis tray was filled with silicone oil. Filter paper moisten with water was positioned at both ends of the gel strips impregnated with the sample, and the gel strips were set in the electrophoresis tray such that the gel strips were covered with silicone oil. Electrodes were placed on the gel strips with the filter paper intervening therebetween.

[0456] The maximum current of the isoelectric focusing system was set to 75 .mu.A per gel strip, and the first-dimensional isoelectric focusing was carried out according to the following voltage program: (1) a constant voltage step was performed at a constant voltage of 300 V until the volt-hours reached 750 Vhr (the current variation width during electrophoresis for 30 minutes before the end of this step was 5 .mu.A); (2) the voltage was increased gradually to 1000 V for 300 Vhr; (3) the voltage was further increased gradually to 5000 V for 4500 Vhr; and then (4) the voltage was held at a constant voltage of 5000 V until the total Vhr reached 12000.

[0457] SDS Equilibration of Isoelectric Focusing Gels

[0458] After the aforementioned first-dimensional isoelectric focusing was done, the gel strips were taken out of the isoelectric focusing system, immersed in an equilibration buffer containing a reducing agent, and shaken at room temperature for 15 minutes. The constituents of the equilibration buffer containing the reducing agent are as mentioned below.

[0459] 100 mM Tris-HCl (pH 8.0)

[0460] 6M urea

[0461] 30% (v/v) glycerol

[0462] 2% (w/v) SDS

[0463] 1% (w/v) DTT

[0464] Next, the equilibration buffer containing the reducing agent was removed, and then the gel strips were immersed in an equilibration buffer containing an alkylating agent and shaken at room temperature for 15 minutes to obtain SDS-equilibrated gels. The constituents of the equilibration buffer containing the alkylating agent are as mentioned below.

[0465] 100 mM Tris-HCl (pH 8.0)

[0466] 6M urea

[0467] 30% (v/v) glycerol

[0468] 2% (w/v) SDS

[0469] 2.5% (w/v) iodoacetamide

[0470] Second-Dimensional SDS-PAGE

[0471] In this example, the XCell SureLock Mini-Cell electrophoresis system produced by Life Technologies was used. The second-dimensional electrophoresis gels used were NuPAGE 4-12% Bis-Tris Gels produced by Life Technologies. Also, an electrophoresis buffer composed of the following constituents was prepared and used.

[0472] 50 mM MOPS

[0473] 50 mM Tris base

[0474] 0.1% (w/v) SDS

[0475] 1 mM EDTA

[0476] Further, an agarose solution for gel adhesion was used in this example, which was prepared by dissolving 0.5% (w/v) Agarose S (produced by Nippon Gene Co., Ltd.) and a moderate amount of BPB (bromophenol blue) in the electrophoresis buffer.

[0477] SDS-PAGE wells were washed well with the electrophoresis buffer, and then the buffer used for the washing was removed. Next, the washed wells were charged with the fully dissolved agarose solution for gel adhesion. Next, the SDS-equilibrated gel strips were immersed in agarose and closely adhered to second-dimensional electrophoresis gels using tweezers. After it was confirmed that agarose was fully fixed with the gels being closely adhered to each other, electrophoresis was performed at a constant voltage of 200 V for about 45 minutes.

[0478] Fluorescent Staining of Gels

[0479] The gels were fluorescently stained with SYPRO Ruby (produced by Life Technologies).

[0480] First, an airtight container to be used was washed well in advance with 98% (v/v) ethanol. The electrophoresed second-dimensional electrophoresis gel strips were taken out of the SDS-PAGE system, placed onto the washed airtight container, and treated twice by immersion in 50% (v/v) methanol and 7% (v/v) aqueous solution containing acetic acid for 30 minutes. Then, a further immersion treatment was done for 10 minutes, with the solution being replaced by water. Next, the second-dimensional electrophoresis gel strips were immersed in 40 mL of SYPRO Ruby and shaken at room temperature overnight. Thereafter, the SYPRO Ruby was removed, and then the second dimensional electrophoresis gel strips were washed with water and shaken in 10% (v/v) methanol and 7% (v/v) aqueous solution containing acetic acid for 30 minutes. Further shaking was done for at least 30 minutes, with the solution being replaced by water.

[0481] Analysis

[0482] The second-dimensional electrophoresis gels obtained through the foregoing series of treatments were subjected to fluorescent image scanning on Typhoon 9500 (produced by GE). The left panel of FIG. 1A illustrates the result of two-dimensional electrophoresis of proteins contained in flesh of salmon. Molecular weight marker bands are found at the left of the panels. The positions of the bands indicate particular molecular weights (KDa).

Example 2: Identification of Antigens by Immunoblotting (1)

[0483] Identification of antigens by immunoblotting was carried out by taking all the steps up to the step of "Second-dimensional SDS-PAGE" as described above in Example 1, followed by the steps of "Transfer to membrane", "Immunoblotting" and "Analysis" as described below.

[0484] Transfer to Membrane

[0485] Transfer to membrane was done using the following transfer system and transfer buffer. [0486] Transfer system: XCell SureLock Mini-Cell and XCell II Blot Module (produced by Life Technologies) [0487] Transfer buffer: NuPAGE Transfer Buffer (.times.20) (produced by Life Technologies), used in a form diluted 20-fold with milliQ water.

[0488] To be specific, proteins in the second-dimensional electrophoresis gels were transferred to a membrane (PVDF membrane) according to the following procedure. [0489] (1) The PVDF membrane was immersed in 100% methanol followed by milliQ water, and then moved into the transfer buffer to hydrophilize the PVDF membrane. [0490] (2) After sponge, filter paper, the second-dimensional electrophoresis gels prepared by second-dimensional SDS-PAGE, the hydrophilized PVDF membrane, filter paper, and sponge were put in place in this order, the transfer system was energized at a constant voltage of 30 V for one hour.

[0491] Immunoblotting

[0492] Immunoblotting of the membrane was carried out using, as a primary antibody, a serum sample from a patient 1 with a fish allergy or a serum sample from a non-fish-allergic subject. This Fish-allergic patient 1 is a patient diagnosed as immediate-type allergy to fish. This patient developed allergic symptoms to salmon, horse mackerel, sea eel, black porgy, mackerel, sea bream, cod, and young yellowtail and was positive in a prick test.

[0493] Immunoblotting of the membrane was carried out according to the following procedure. [0494] (1) The transferred membrane was shaken in a 5% skim milk/PBST solution (a PBS buffer containing 0.1% Tween 20 nonionic surfactant) at room temperature for one hour. [0495] (2) The membrane was left to stand in a solution of 5% serum as a primary antibody in 5% skim milk/PBST at room temperature for one hour. [0496] (3) The membrane was washed with a PBST solution (5 min..times.3 times). [0497] (4) The membrane was left to stand in a solution of 1:5000 dilution of the secondary antibody, anti-human IgE-HRP (horseradish peroxidase), with a 5% skim milk/PBST solution at room temperature for one hour. [0498] (5) The membrane was washed with a PBST solution (5 min..times.3 times). [0499] (6) The membrane was left to stand in Pierce Western Blotting Substrate Plus (produced by Thermo Fisher Scientific) for 5 minutes.

[0500] Analysis

[0501] The membrane obtained through the foregoing series of treatments was subjected to fluorescent image scanning on Typhoon 9500 (produced by GE).

[0502] The immunoblot obtained with the serum from Fish-allergic patient 1 was compared with that obtained with the control serum from Non-fish-allergic subject. Nine spots that are different from those with the serum of Non-fish-allergic subject (FIG. 3) and different from the known salmon allergen proteins were detected on an immunoblot of the proteins contained in flesh of salmon with the serum of Fish-allergic patient 1 (FIG. 1A, right panel). The detected spots are indicated in the immunoblot (FIG. 1A, right panel).

[0503] The molecular weights and isoelectric points of the 9 spots are as follows. [0504] Spot 1: Molecular weight 90 to 120 kDa, pI 3.0 to 6.0 [0505] Spot 2: Molecular weight 120 to 160 kDa, pI 4.0 to 7.0 [0506] Spot 3: Molecular weight 90 to 120 kDa, pI 5.0 to 8.0 [0507] Spot 4: Molecular weight 90 to 120 kDa, pI 6.0 to 8.0 [0508] Spot 5: Molecular weight 60 to 90 kDa, pI 3.0 to 6.0 [0509] Spot 6: Molecular weight 70 to 100 kDa, pI 5.0 to 7.0 [0510] Spot 7: Molecular weight 40 to 70 kDa, pI 4.0 to 7.0 [0511] Spot 8: Molecular weight 40 to 70 kDa, pI 4.0 to 7.0 [0512] Spot 9: Molecular weight 20 to 50 kDa, pI 4.0 to7.0

Example 3: Mass Spectrometry and Identification of Antigens (1)

[0513] The amino acid sequences of the antigens that form the above nine protein spots were identified by mass spectroscopy.

[0514] To be specific, protein extraction and mass spectroscopy were done by the following procedure. [0515] (1) Flesh of salmon was subjected to protein extraction, two-dimensional electrophoresis and transfer to membrane by following the procedures described in Example 1 and 2, and the resulting membrane was stained by shaking in a solution of 0.008% Direct blue in 40% ethanol and 10% acetic acid. [0516] (2) Then, the membrane was decolorized by repeating a 5-minute treatment with 40% ethanol and 10% acetic acid three times, washed with water for 5 minutes, and then dried by air. [0517] (3) A protein spot of interest was cut out with a clean cutter blade and put into a centrifugal tube. The cut membrane was subjected to hydrophilization with 50 .mu.L of methanol, followed by washing with 100 .mu.L of water twice and then centrifugal cleaning. Thereafter, 20 .mu.L of 20 mM NH.sub.4HCO.sub.3 and 50% acetonitrile were added. [0518] (4) 1 .mu.L of 1 pmol/.mu.L lysyl endopeptidase (produced by WAKO) was added, and the solution was left to stand at 37.degree. C. for 60 minutes and then collected in a new centrifugal tube. After 20 .mu.L of 20 mM NH.sub.4HCO.sub.3 and 70% acetonitrile were added to the membrane, the membrane was immersed therein at room temperature for 10 minutes, and the resulting solution was further collected. The solution was dissolved with 0.1% formic acid and 10 .mu.L of 4% acetonitrile and transferred to a tube. [0519] (5) The collected solution was dried under reduced pressure, dissolved with 15 .mu.L of solution A (a 0.1% formic acid/4% acetonitrile solution), and analyzed by mass spectroscopy (ESI-TOF6600, produced by AB Sciex). [0520] (6) Identification of proteins based on the MS data obtained with the mass spectrometer was done by searching NCBI or UniProt.

[0521] Results

[0522] The mass spectrometry of the spots resulted in the detection of the following amino acid sequences. [0523] Spot 1: the amino acid sequences set forth in SEQ ID NO: 3 [0524] Spot 2: the amino acid sequences set forth in SEQ ID NO: 6 to 8 [0525] Spot 3: the amino acid sequences set forth in SEQ ID NO: 11 to 17 [0526] Spot 4: the amino acid sequences set forth in SEQ ID NO: 20 to 24 [0527] Spot 5: the amino acid sequences set forth in SEQ ID NO: 27 to 31 [0528] Spot 6: the amino acid sequences set forth in SEQ ID NO: 34 to 41 [0529] Spot 7: the amino acid sequences set forth in SEQ ID NO: 44 to 54 [0530] Spot 8: the amino acid sequences set forth in SEQ ID NO: 57 to 59 [0531] Spot 9: the amino acid sequences set forth in SEQ ID NO: 62 to 68

[0532] Furthermore, the spots were identified as the following proteins by the analysis of the MS data obtained from the mass spectrometer for spot 1 at NCBI and for spots 2 to 9 with UniProt. [0533] Spot 1: Alpha-actinin-3 (NCBI protein accession number XP_014051545.1, DNA accession number XM_014196070.1) (amino acid sequence: SEQ ID NO: 2, encoding nucleotide sequence: SEQ ID NO: 1) [0534] Spot 2: EEF1A2 binding protein-like (UniProt protein accession number B5RI29, DNA accession number ACH85270.1) (amino acid sequence: SEQ ID NO: 5, encoding nucleotide sequence: SEQ ID NO: 4) [0535] Spot 3: Alpha-1,4-glucan phosphorylase (UniProt protein accession number B5DG55, DNA accession number ACH70729.1) (amino acid sequence: SEQ ID NO: 10, encoding nucleotide sequence: SEQ ID NO: 9) [0536] Spot 4: Elongation factor 2 (UniProt protein accession number C0H9N2, DNA accession number ACN10751.1) (amino acid sequence: SEQ ID NO: 19, encoding nucleotide sequence: SEQ ID NO: 18) [0537] Spot 5: Heat shock cognate 70 kDa protein (UniProt protein accession number B5X3U6, DNA accession number ACI33977.1) (amino acid sequence: SEQ ID NO: 26, encoding nucleotide sequence: SEQ ID NO: 25) [0538] Spot 6: Serotransferrin (UniProt protein accession number P79815, DNA accession number BAA13759.1) (amino acid sequence: SEQ ID NO: 33, encoding nucleotide sequence: SEQ ID NO: 32) [0539] Spot 7: Myosin binding protein H-like (UniProt protein accession number B5DG45, DNA accession number ACH70719.1) (amino acid sequence: SEQ ID NO: 43, encoding nucleotide sequence: SEQ ID NO: 42) [0540] Spot 8: Desmin (Fragment) (UniProt protein accession number Q8UWF1, DNA accession number CAC83053.1) (amino acid sequence: SEQ ID NO: 56, encoding nucleotide sequence: SEQ ID NO: 55) [0541] Spot 9: Capping protein (Actin filament) muscle Z-line beta (UniProt protein accession number B5DFX6, DNA accession number ACH70650.1) (amino acid sequence: SEQ ID NO: 61, encoding nucleotide sequence: SEQ ID NO: 60)

Example 4: Confirmation of Antigens in Other Fish Species (1)

[0542] Using flesh of the 9 fish species, horse mackerel, sea eel, black porgy, mackerel, sea bream, cod, young yellowtail, eel, and flatfish, other than salmon, antigens were confirmed by the same procedure as the procedure described in Example 1 and Example 2.

[0543] Immunoblots obtained with the serum from Fish-allergic patient 1 was compared with that obtained with the control serum from Non-fish-allergic subject. Spots that are different from those with the serum from Non-fish-allergic subject (FIG. 3) and correspond to a part of the 9 spots obtained in salmon were detected on immunoblots (FIG. 2) of proteins contained in flesh of each of the 9 fish species stained with the serum from Fish-allergic patient 1.

[0544] Spots detected in each fish species are as follows. [0545] Horse mackerel: Spots 1, 3-5, 8, and 9 [0546] Sea eel: Spots 1, 3, 5, and 9 [0547] Black porgy: Spots 1-6, 8, and 9 [0548] Mackerel: Spots 1, 3, 5, 8, and 9 [0549] Sea bream: Spots 1, 3-6, 8, and 9 [0550] Cod: Spots 1, 3, 5, 7-9 [0551] Young yellowtail: Spots 1, 3-5, 8, 9 [0552] Eel: Spots 1, 3, 4, 6, 9 [0553] Flatfish: Spots 1, 4, 6

Example 5: Confirmation of Antigens by Immunoblotting (2)

[0554] An immunoblot obtained with serum from a fish-allergic patient was compared with that obtained with the control serum from Non-fish-allergic subject in the same way as in Example 2. Three new spots (Spots 11, 12, 14) that are different from those with the serum from Non-fish-allergic subject (FIG. 3) and different from the known salmon allergen proteins, other than 7 (spots 1-6, 9) of the 9 spots detected in Example 2, were detected on an immunoblot (FIG. 1B, left panel) of proteins contained in flesh of salmon stained with the serum from Fish-allergic patient 2. The total 10 spots detected are indicated in the immunoblot (FIG. 1B, left panel).

[0555] The molecular weights and isoelectric points of the 3 new spots detected are as follows. [0556] Spot 11: Molecular weight 90 to 110, pI 6.5 to 7.0 [0557] Spot 12: Molecular weight 120 to 140, pI 5.0 to 6.0 [0558] Spot 14: Molecular weight 30 to 40, pI 6.5 to 7.5

Example 6: Mass Spectrometry and Identification of Antigens (2)

[0559] The amino acid sequences of the antigens that form the 3 spots newly detected were identified by mass spectroscopy in the same way as in Example 3.

[0560] The mass spectrometry of the spots resulted in the detection of the following amino acid sequences. [0561] Spot 11: Amino acid sequences set forth in SEQ ID NOs: 111 to 119 [0562] Spot 12: Amino acid sequences set forth in SEQ ID NOs: 122 to 136 [0563] Spot 14: Amino acid sequences set forth in SEQ ID NOs: 145 to 149

[0564] Furthermore, the spots were identified as the following proteins by the analysis of the MS data obtained from the mass spectrometer for the spots at NCBI. [0565] Spot 11: Glycogen phosphorylase, muscle form-like (NCBI protein accession number XP_013984904.1, DNA accession number XM_014129429.1) (amino acid sequence: SEQ ID NO: 110, encoding nucleotide sequence: SEQ ID NO: 109) [0566] Spot 12: Myosin-binding protein C, fast-type-like (NCBI protein accession number XP_014014310.1, DNA accession number XM_014158835.1) (amino acid sequence: SEQ ID NO: 121, encoding nucleotide sequence: SEQ ID NO: 120) [0567] Spot 14: L-lactate dehydrogenase A chain-like (NCBI protein accession number XP_014003141.1, DNA accession number XM_014147666.1) (amino acid sequence: SEQ ID NO: 144, encoding nucleotide sequence: SEQ ID NO: 143)

Example 7: Confirmation of Antigens by Immunoblotting (3)

[0568] An immunoblot obtained with the serum from a fish-allergic patient was compared with that obtained with the control serum from Non-fish-allergic subject, in the same way as in Example 2. Two new spots (Spots 10, 13) that are different from those with the serum from Non-fish-allergic subject (FIG. 3) and different from the known salmon allergen proteins, other than 2 (Spots 7, 8) of the 9 spots detected in Example 2, were detected on an immunoblot (FIG. 1B, right panel) of proteins contained in flesh of salmon stained with the serum from Fish-allergic patient 3. The total 4 spots detected are indicated in the immunoblot (FIG. 1B, right panel).

[0569] The molecular weights and isoelectric points of the 2 new spots detected are as follows. [0570] Spot 10: Molecular weight 200 to 230, pI 4.5 to 5.5 [0571] Spot 13: Molecular weight 45 to 55, pI 4.0 to 5.5

Example 8: Mass Spectrometry and Identification of Antigens (3)

[0572] The amino acid sequences of the antigens that form the 2 spots newly detected were identified by mass spectroscopy in the same way as in Example 3.

[0573] The mass spectrometry of the spots resulted in the detection of the following amino acid sequences. [0574] Spot 10: Amino acid sequences set forth in SEQ ID NOs: 71 to 108 [0575] Spot 13: Amino acid sequences set forth in SEQ ID NOs: 139 to 142

[0576] Furthermore, the spots were identified as the following proteins by the analysis of the MS data obtained from the mass spectrometer for the spots at NCBI. [0577] Spot 10: Myosin heavy chain, fast skeletal muscle-like (NCBI protein accession number XP_014039990.1, DNA accession number XM_014184515.1) (amino acid sequence: SEQ ID NO: 70, encoding nucleotide sequence: SEQ ID NO: 69) [0578] Spot 13: ATP synthase subunit beta, mitochondrial (NCBI protein accession number XP_014007238.1, DNA accession number XM_014151763.1) (amino acid sequence: SEQ ID NO: 138, encoding nucleotide sequence: SEQ ID NO: 137)

Example 9: Confirmation of Antigens in Other Fish Species (2)

[0579] Using flesh of the 6 fish species, horse mackerel, sea eel, salmon, mackerel, sea bream, and cod, the antigen was confirmed in the same way as in Example 4. Immunoblots obtained with the serum from Fish-allergic patient 4 was compared with that obtained with the control serum from Non-fish-allergic subject. Spots that are different from those with the serum from Non-fish-allergic subject (FIG. 3) and correspond to a part of the 14 spots obtained in salmon with the serum from Fish-allergic patients 1 to 3 were detected on immunoblots (FIG. 4) of proteins contained in flesh of each of the 6 fish species stained with the serum from Fish-allergic patient 4.

[0580] Spots detected in each fish species are as follows. [0581] Horse mackerel: Spots 1, 3, 4, 8, 10, 11, and 14 [0582] Sea eel: Spots 1, 4, 10, and 11 [0583] Salmon: Spots 2, 8, and 10-14 [0584] Mackerel: Spots 4, 8, 10, 11, 13, and 14 [0585] Sea bream: Spots 1, 4, 6, 8, 10, and 13 [0586] Cod: Spots 1, 5, 8, 10, 11, and 13

INDUSTRIAL APPLICABILITY

[0587] The present invention can provide novel antigens of an allergy to fish, methods and kits for diagnosing an allergy to fish, a pharmaceutical composition comprising the antigen, and fish or fish eggs in which the antigen is eliminated or reduced, a processed product of the fish or fish eggs, or fish which lay the fish eggs or have hatched from the eggs. The present invention can further provide a tester for determining the presence or absence of a fish antigen in an object of interest.

Sequence CWU 1

1

14912685DNAArtificial SequencePREDICTED alpha-actinin-3 1atgacggcaa tcgaaactca ggtgcaatat ggctcctaca tgacttcgga gcaagtctac 60atgacccaag aggatgattg ggacagggac cttctgttgg accctgcctg ggagaaacag 120cagcgcaaga ccttcaccgc ctggtgcaac tctcacctgc gtaaagcggg cacacagatt 180gagaacattg aggaggactt caggaatggg ctcaagctca tgttgctgtt agaggtcatc 240tcaggtgaaa ggcttcccaa accagacaaa ggcaaaatgc gtttccacaa aatcgccaac 300gtgaacaagg ccctggattt catctgcagc aagggagtca agctggtgtc catcggtgcc 360gaggagattg tggatggtaa tgtgaagatg accctgggga tgatctggac catcattctg 420cgtttcgcca tccaggacat ctctgtagag gagacctctg ctaaggaggg tctgttgctg 480tggtgccaga ggaagactgc cccctacagg aatgtcaatg tgcagaactt ccacatcagt 540tggaaggatg gcctggcact gtgtgccctc atccacagac acagacctga cctcattgac 600tactccaaac tgcgcaagga tgaccccatg ggcaatctca acactgcctt tgaggtggca 660gagaagtacc tggacatccc caagatgttg gatgcagaag atattgtgaa caccccgaag 720cccgacgaga aagccatcat gacctatgtg tcctgcttct atcatgcctt cgctggagcc 780gagcaggccg agacagctgc caataggatc tgcaaggtgt tggctgtcaa ccaggagaac 840gagaagctca tggaggagta tgagaagctg gccagtgagc tgctggagtg gatccgtcgc 900accatcccct ggctggagaa ccgcgtggct gagcagacca tgcgcgccat gcagcagaag 960ctggaggact tccgtgacta ccgtcgcgtc cacaagcctc ccaaggtgca ggagaagtgt 1020cagctggaga ttaacttcaa caccctgcag accaagctga ggctgagcaa caggcccgcc 1080ttcatgccct ccgagggcaa gatggtgtcg gacattgcca atgcctggaa gggtctggag 1140caggtagaga agggctatga ggagtggctg ctcactgaga tcagacgcct ggagaggctg 1200gaccacctgg ctgagaagtt caagcagaag tcttccctgc atgagtcctg gacctcaggt 1260aaggtggagc tgctgtccat gaaggactat gagtctgcct cactgatgga gatccgtgcc 1320ctgatgagga agcacgaggc gtttgagagc gacctggctg cccaccagga cagagtggag 1380cagattgctg ccatcgccca ggagctcaat gagctggact atcatgatgc cgtcaccatc 1440aacgcccgct gccagggtat ttgtgaccag tgggacaacc tgggcacctt gacccagaag 1500aggagagact cactggagcg tgtggagaag ctgtgggaga ccatcgacca gctgtacctg 1560gagtttgcca agagagcagc ccccttcaac aactggatgg acggagccat ggaggactta 1620caggacatgt tcattgtgca cagcattgag gagatccaga gtctgatcac agcccatgac 1680cagttcaagg ccaccctgcc agaggcagat aaggagcgcg ttgcaaccat gggaatccag 1740aatgagatcg tgaagatcgc tcagacctac ggcatcaagc tgtcaggagt caacccctac 1800accaaccttt ccccccagga catcaccgac aaatgggatg ctgtgaaaca cctggtgccc 1860ctcagggatc agatgctgca ggaggaggtg gccaggcagc agtccaacga gaggctgagg 1920cgccagttcg ctgcccaggc caacatcatc ggaccctgga tccagaccaa gatggaggag 1980atcagccatg tgtctgtgga catcgccggc tccctggagg aacagatgag caacctgaag 2040cagtacgagc agaacatcat caactacaag tgcaacatcg acaagctgga gggagaccac 2100caactcagcc aggagtccct catctttgac aacaagcaca ccaactacac catggagcat 2160gtgcgtgtgg gctgggagca gctcctcacc accatcgccc gcaccatcaa cgaggtggag 2220aaccagatcc tgacccgaga tgccaagggc atcagccagg agcagctcaa cgagttcaga 2280gcctccttca accactttga caggaagaga aatggtatga tggacccaga tgacttccgt 2340gcctgtctca tctccatggg ctacgatctg ggtgaggtgg agtttgcccg catcatgacg 2400ctggtggatt ccaacaacac aggtgtggtg accttccagg ccttcatcga cttcatgacc 2460cgcgagacgg ccgagacaga caccgccgat caggtcatgg cctcattcaa gatcctggcc 2520tctgacaaga catacatcac agtggaagaa ctgcgcaggg agctgccccc agagcaggcc 2580gactactgca tcagccgcat gaccagttac atcggcagcg gcgcaccccc aggcgccctg 2640gactacatct ccttctccag tgccctgtac ggagagagcg actta 26852895PRTArtificial SequencePREDICTED alpha-actinin-3 2Met Thr Ala Ile Glu Thr Gln Val Gln Tyr Gly Ser Tyr Met Thr Ser1 5 10 15Glu Gln Val Tyr Met Thr Gln Glu Asp Asp Trp Asp Arg Asp Leu Leu 20 25 30Leu Asp Pro Ala Trp Glu Lys Gln Gln Arg Lys Thr Phe Thr Ala Trp 35 40 45Cys Asn Ser His Leu Arg Lys Ala Gly Thr Gln Ile Glu Asn Ile Glu 50 55 60Glu Asp Phe Arg Asn Gly Leu Lys Leu Met Leu Leu Leu Glu Val Ile65 70 75 80Ser Gly Glu Arg Leu Pro Lys Pro Asp Lys Gly Lys Met Arg Phe His 85 90 95Lys Ile Ala Asn Val Asn Lys Ala Leu Asp Phe Ile Cys Ser Lys Gly 100 105 110Val Lys Leu Val Ser Ile Gly Ala Glu Glu Ile Val Asp Gly Asn Val 115 120 125Lys Met Thr Leu Gly Met Ile Trp Thr Ile Ile Leu Arg Phe Ala Ile 130 135 140Gln Asp Ile Ser Val Glu Glu Thr Ser Ala Lys Glu Gly Leu Leu Leu145 150 155 160Trp Cys Gln Arg Lys Thr Ala Pro Tyr Arg Asn Val Asn Val Gln Asn 165 170 175Phe His Ile Ser Trp Lys Asp Gly Leu Ala Leu Cys Ala Leu Ile His 180 185 190Arg His Arg Pro Asp Leu Ile Asp Tyr Ser Lys Leu Arg Lys Asp Asp 195 200 205Pro Met Gly Asn Leu Asn Thr Ala Phe Glu Val Ala Glu Lys Tyr Leu 210 215 220Asp Ile Pro Lys Met Leu Asp Ala Glu Asp Ile Val Asn Thr Pro Lys225 230 235 240Pro Asp Glu Lys Ala Ile Met Thr Tyr Val Ser Cys Phe Tyr His Ala 245 250 255Phe Ala Gly Ala Glu Gln Ala Glu Thr Ala Ala Asn Arg Ile Cys Lys 260 265 270Val Leu Ala Val Asn Gln Glu Asn Glu Lys Leu Met Glu Glu Tyr Glu 275 280 285Lys Leu Ala Ser Glu Leu Leu Glu Trp Ile Arg Arg Thr Ile Pro Trp 290 295 300Leu Glu Asn Arg Val Ala Glu Gln Thr Met Arg Ala Met Gln Gln Lys305 310 315 320Leu Glu Asp Phe Arg Asp Tyr Arg Arg Val His Lys Pro Pro Lys Val 325 330 335Gln Glu Lys Cys Gln Leu Glu Ile Asn Phe Asn Thr Leu Gln Thr Lys 340 345 350Leu Arg Leu Ser Asn Arg Pro Ala Phe Met Pro Ser Glu Gly Lys Met 355 360 365Val Ser Asp Ile Ala Asn Ala Trp Lys Gly Leu Glu Gln Val Glu Lys 370 375 380Gly Tyr Glu Glu Trp Leu Leu Thr Glu Ile Arg Arg Leu Glu Arg Leu385 390 395 400Asp His Leu Ala Glu Lys Phe Lys Gln Lys Ser Ser Leu His Glu Ser 405 410 415Trp Thr Ser Gly Lys Val Glu Leu Leu Ser Met Lys Asp Tyr Glu Ser 420 425 430Ala Ser Leu Met Glu Ile Arg Ala Leu Met Arg Lys His Glu Ala Phe 435 440 445Glu Ser Asp Leu Ala Ala His Gln Asp Arg Val Glu Gln Ile Ala Ala 450 455 460Ile Ala Gln Glu Leu Asn Glu Leu Asp Tyr His Asp Ala Val Thr Ile465 470 475 480Asn Ala Arg Cys Gln Gly Ile Cys Asp Gln Trp Asp Asn Leu Gly Thr 485 490 495Leu Thr Gln Lys Arg Arg Asp Ser Leu Glu Arg Val Glu Lys Leu Trp 500 505 510Glu Thr Ile Asp Gln Leu Tyr Leu Glu Phe Ala Lys Arg Ala Ala Pro 515 520 525Phe Asn Asn Trp Met Asp Gly Ala Met Glu Asp Leu Gln Asp Met Phe 530 535 540Ile Val His Ser Ile Glu Glu Ile Gln Ser Leu Ile Thr Ala His Asp545 550 555 560Gln Phe Lys Ala Thr Leu Pro Glu Ala Asp Lys Glu Arg Val Ala Thr 565 570 575Met Gly Ile Gln Asn Glu Ile Val Lys Ile Ala Gln Thr Tyr Gly Ile 580 585 590Lys Leu Ser Gly Val Asn Pro Tyr Thr Asn Leu Ser Pro Gln Asp Ile 595 600 605Thr Asp Lys Trp Asp Ala Val Lys His Leu Val Pro Leu Arg Asp Gln 610 615 620Met Leu Gln Glu Glu Val Ala Arg Gln Gln Ser Asn Glu Arg Leu Arg625 630 635 640Arg Gln Phe Ala Ala Gln Ala Asn Ile Ile Gly Pro Trp Ile Gln Thr 645 650 655Lys Met Glu Glu Ile Ser His Val Ser Val Asp Ile Ala Gly Ser Leu 660 665 670Glu Glu Gln Met Ser Asn Leu Lys Gln Tyr Glu Gln Asn Ile Ile Asn 675 680 685Tyr Lys Cys Asn Ile Asp Lys Leu Glu Gly Asp His Gln Leu Ser Gln 690 695 700Glu Ser Leu Ile Phe Asp Asn Lys His Thr Asn Tyr Thr Met Glu His705 710 715 720Val Arg Val Gly Trp Glu Gln Leu Leu Thr Thr Ile Ala Arg Thr Ile 725 730 735Asn Glu Val Glu Asn Gln Ile Leu Thr Arg Asp Ala Lys Gly Ile Ser 740 745 750Gln Glu Gln Leu Asn Glu Phe Arg Ala Ser Phe Asn His Phe Asp Arg 755 760 765Lys Arg Asn Gly Met Met Asp Pro Asp Asp Phe Arg Ala Cys Leu Ile 770 775 780Ser Met Gly Tyr Asp Leu Gly Glu Val Glu Phe Ala Arg Ile Met Thr785 790 795 800Leu Val Asp Ser Asn Asn Thr Gly Val Val Thr Phe Gln Ala Phe Ile 805 810 815Asp Phe Met Thr Arg Glu Thr Ala Glu Thr Asp Thr Ala Asp Gln Val 820 825 830Met Ala Ser Phe Lys Ile Leu Ala Ser Asp Lys Thr Tyr Ile Thr Val 835 840 845Glu Glu Leu Arg Arg Glu Leu Pro Pro Glu Gln Ala Asp Tyr Cys Ile 850 855 860Ser Arg Met Thr Ser Tyr Ile Gly Ser Gly Ala Pro Pro Gly Ala Leu865 870 875 880Asp Tyr Ile Ser Phe Ser Ser Ala Leu Tyr Gly Glu Ser Asp Leu 885 890 895314PRTArtificial SequencePREDICTED alpha-actinin-3 fragment 3Glu Arg Val Ala Thr Met Gly Ile Gln Asn Glu Ile Val Lys1 5 1043591DNAArtificial SequenceEEF1A2 binding protein-like 4atgtggaaaa agaaatcaaa gctcacggac cagacggcca ctggccaagt tgggatcaag 60aagagatcaa aagtccctgg agttatgatc acgcagtatg tggagaaaat accagatggg 120aaaagccacc ctgacttcac ccgcaagcct atcgcgttga ccattcagga gggtaaattc 180gccttcttca aagccctggt tattggagat ccagaaccaa ccgtgacatg gggcagaaat 240aatggagatg tgtcagatac atcaaaatat gtgacaaaat atgaccctgc tacacgtgag 300cacttatttg agatggccaa tgtaaaacca gaacaagcag acacctacaa atgctttgca 360gctaatgagt ttggaagagc agtggtcaca gtggtcctca atgttattga agttgggttc 420aagaaagcac aagcggactc acaggtgcaa ccagaggcag ctgttgcaga ttttaaatct 480gtattgaaga gaaaaagtaa aattcagccc aaaatggaaa agaaagaaga tggagaaata 540gatccaagat tttgggaact cttgataagt gctgacaaga aagactatga gagcctcatg 600ttggagtttg gagtcactga cttccgcttt atgctgaaga cactgaatga gattaagaag 660gaaagagagg aagagcaagc acagttcatt gaaaacctag ctaacctgaa acctattgaa 720gttggacccg atggctgtgc aaccttttca atagacatgg atctcattga acaaagcagc 780aggatcttcc tctacaagga tggagtgatg attccataca gcaaggagtt gggagataca 840atcaaacaca gcctaaagat agtgggccga aagtatcagt tcagcataag ggatctgttt 900cctgatgatg ctgggctcta ccaggtggat gttgaggacg taaatgtatt ctccaccgat 960tttaagattc ccatggtgga cttcctggtc aagattcagg agtgtaaggc catggagaga 1020gaggatgctg tgtttgagtg tgtcctgtca cagcccttcg gcaagatcat gtgggttggc 1080aagaacttac cattggaggc aggggataaa tatgatattg aggtttcaga agacaagctc 1140atccacagac taatcattaa agacgttgct atggtggaca aaggcatcta tgccgctgtg 1200gcaggaatca aatcttgcaa tgcctttctt gtggttgaag ccgacaaggg tgaacccggc 1260aaaaagaaac aacgtaaaac cacaagggca ggaggagctg gagttgacct gacggcgatt 1320gcccaagagc aggcagttaa aaacacggca gacagagagg tgctgaagga gaaggtgaaa 1380gcaatcaatg acgagagagc ggctaatgcc actgcagcac ctgagacttc agctgaagct 1440aaagctaaag ttaaggtggt agaggcttcc caaacaggag ctccaaaaca gggaccggca 1500gttaaagggt ctgaccataa atccgtggat aatgagggat acagcattaa gagtggactc 1560tcagatgtct ttgctctccg tggcaagaaa ggtgaactgg tttgtgagat gagccatgaa 1620gttgatgggg cctggttcaa ggatggagag aagttatcca ccacagatgg aatagccata 1680gtgaaagatg gaacgagaca cacgatgacc attcatagca gtagtgaaga cgacactgga 1740gtctatcaca ttgaagcggg gggatttaaa tcagaggcaa aagtcactgt gggagaatta 1800cctgccattg atgctgatga cctccacaag ttttctaagc ctgtgacaat aaaagtgggc 1860cagaatgcat cctggaagat gccttataca ccacaggaca acttggaggt gaaatggttt 1920aaggatggca aagagttgaa ggatggtggt ggggtgaagt tggtgaagga ggtcaagcac 1980agccggctgc tgctccggga gtgtctgcgt tctgacgctg gggagatcaa gattcaactc 2040aaaaacccct ttggcactat agaggccaca tctcgactga ttgtccttga caagcccggc 2100ccaccagaag gcccggtgga aatcttggag accacctcca ctgtgattga gctgcagtgg 2160ggcgctccta aggacgacgg tggctccccg gtgactaact acatcattga gcgccagcag 2220ctgggacaga ccgtgtggaa gaagatgggt gatgtcgcag ctgacaaaac cacctacagg 2280gacaggaatg tggtccatgg gaaactgtac atctacaata tctacgcagt gaacccagag 2340gggaccagtg atgcactgca gactgaggaa acaatggctg gcgtattgat atttgctggc 2400cgacctggag caccaaaagt ggtcagcgca tcaaagacct gcatcaacct gaaatgggag 2460cccccagaag atgacggagg aattaagatt gatggctatc aactggaaaa acgcaaaaag 2520gacacagctc agtggattgc tttgaaccca gtaactgagc ctattgaagt gctggagtac 2580gcagtgaagg atgttgttga gggggcggag tatgagttca gggtatcggc catcaacgtt 2640tctggggcgg gagagttcag tctcccctct gtgatggtga ctgcaaagaa tcccaacatg 2700aggcctacat tcaaagatcc agaggacttc atggtgatca gggcgggaaa ctctgtgaga 2760atcaaagttc tctatgaggc tgagcctcca ccggagatca cttggatgaa ggacaatgag 2820cctgtatcca gttttataca gatcatcaac acagagggct gttcccagct tgtgatcccc 2880tcaacaaaac gctctgattc aggaaattac accattgtgg ctaagaataa agttggagag 2940gccagctttg acattgaggt cctagtcaca gatgagccaa agcctcctgg tgcagtggag 3000ctggagcaga ttgtccatgg caaagttatt gtgtcctggg aggcctctcc agaccaggag 3060ctggacaaca ggctgtatta catggtggcc gagcacgact ccagcacacg catgtggcac 3120actgtggcag accgcatctt tgacaattca tacacagcca ataacatcat gcctggaagg 3180gagtaccact tcaaaatcta tgccaagaac gacatgggca tgtcagaccc ctccatgtca 3240cccacctggg gcatcaacag caacagaatt cctataaaca caaacacgcc agtggaagtc 3300agctttgaga aaccaccatc tgtcctggtc cctctgaaga tgcactcgcc accaaaagga 3360ttccagatgt acatgacctg cgccatccgg ggatgcccca cacccagcgt cacgtggcac 3420ctgaacaacg tctgcatcaa tggtgacagc aactactaca tcaccaactc gtatggcgtg 3480tgctccatgt acatccttag ggtcaggccc aaggacgccg gagaatacaa agttgtcgca 3540gttaactcct tcggcaaggc agaatgctcc actaaacttg ttgttaaaga c 359151197PRTArtificial SequenceEEF1A2 binding protein-like 5Met Trp Lys Lys Lys Ser Lys Leu Thr Asp Gln Thr Ala Thr Gly Gln1 5 10 15Val Gly Ile Lys Lys Arg Ser Lys Val Pro Gly Val Met Ile Thr Gln 20 25 30Tyr Val Glu Lys Ile Pro Asp Gly Lys Ser His Pro Asp Phe Thr Arg 35 40 45Lys Pro Ile Ala Leu Thr Ile Gln Glu Gly Lys Phe Ala Phe Phe Lys 50 55 60Ala Leu Val Ile Gly Asp Pro Glu Pro Thr Val Thr Trp Gly Arg Asn65 70 75 80Asn Gly Asp Val Ser Asp Thr Ser Lys Tyr Val Thr Lys Tyr Asp Pro 85 90 95Ala Thr Arg Glu His Leu Phe Glu Met Ala Asn Val Lys Pro Glu Gln 100 105 110Ala Asp Thr Tyr Lys Cys Phe Ala Ala Asn Glu Phe Gly Arg Ala Val 115 120 125Val Thr Val Val Leu Asn Val Ile Glu Val Gly Phe Lys Lys Ala Gln 130 135 140Ala Asp Ser Gln Val Gln Pro Glu Ala Ala Val Ala Asp Phe Lys Ser145 150 155 160Val Leu Lys Arg Lys Ser Lys Ile Gln Pro Lys Met Glu Lys Lys Glu 165 170 175Asp Gly Glu Ile Asp Pro Arg Phe Trp Glu Leu Leu Ile Ser Ala Asp 180 185 190Lys Lys Asp Tyr Glu Ser Leu Met Leu Glu Phe Gly Val Thr Asp Phe 195 200 205Arg Phe Met Leu Lys Thr Leu Asn Glu Ile Lys Lys Glu Arg Glu Glu 210 215 220Glu Gln Ala Gln Phe Ile Glu Asn Leu Ala Asn Leu Lys Pro Ile Glu225 230 235 240Val Gly Pro Asp Gly Cys Ala Thr Phe Ser Ile Asp Met Asp Leu Ile 245 250 255Glu Gln Ser Ser Arg Ile Phe Leu Tyr Lys Asp Gly Val Met Ile Pro 260 265 270Tyr Ser Lys Glu Leu Gly Asp Thr Ile Lys His Ser Leu Lys Ile Val 275 280 285Gly Arg Lys Tyr Gln Phe Ser Ile Arg Asp Leu Phe Pro Asp Asp Ala 290 295 300Gly Leu Tyr Gln Val Asp Val Glu Asp Val Asn Val Phe Ser Thr Asp305 310 315 320Phe Lys Ile Pro Met Val Asp Phe Leu Val Lys Ile Gln Glu Cys Lys 325 330 335Ala Met Glu Arg Glu Asp Ala Val Phe Glu Cys Val Leu Ser Gln Pro 340 345 350Phe Gly Lys Ile Met Trp Val Gly Lys Asn Leu Pro Leu Glu Ala Gly 355 360 365Asp Lys Tyr Asp Ile Glu Val Ser Glu Asp Lys Leu Ile His Arg Leu 370 375 380Ile Ile Lys Asp Val Ala Met Val Asp Lys Gly Ile Tyr Ala Ala Val385 390 395 400Ala Gly Ile Lys Ser Cys Asn Ala Phe Leu Val Val Glu Ala Asp Lys 405 410 415Gly Glu Pro Gly Lys Lys Lys Gln Arg Lys Thr Thr Arg Ala Gly Gly 420 425 430Ala Gly Val Asp Leu Thr Ala Ile Ala Gln Glu Gln Ala Val Lys Asn 435 440 445Thr Ala Asp Arg Glu Val Leu Lys Glu Lys Val Lys Ala Ile Asn Asp 450 455 460Glu Arg Ala Ala Asn Ala Thr Ala Ala Pro Glu Thr Ser Ala Glu Ala465 470 475 480Lys Ala Lys Val Lys Val Val Glu Ala Ser Gln Thr

Gly Ala Pro Lys 485 490 495Gln Gly Pro Ala Val Lys Gly Ser Asp His Lys Ser Val Asp Asn Glu 500 505 510Gly Tyr Ser Ile Lys Ser Gly Leu Ser Asp Val Phe Ala Leu Arg Gly 515 520 525Lys Lys Gly Glu Leu Val Cys Glu Met Ser His Glu Val Asp Gly Ala 530 535 540Trp Phe Lys Asp Gly Glu Lys Leu Ser Thr Thr Asp Gly Ile Ala Ile545 550 555 560Val Lys Asp Gly Thr Arg His Thr Met Thr Ile His Ser Ser Ser Glu 565 570 575Asp Asp Thr Gly Val Tyr His Ile Glu Ala Gly Gly Phe Lys Ser Glu 580 585 590Ala Lys Val Thr Val Gly Glu Leu Pro Ala Ile Asp Ala Asp Asp Leu 595 600 605His Lys Phe Ser Lys Pro Val Thr Ile Lys Val Gly Gln Asn Ala Ser 610 615 620Trp Lys Met Pro Tyr Thr Pro Gln Asp Asn Leu Glu Val Lys Trp Phe625 630 635 640Lys Asp Gly Lys Glu Leu Lys Asp Gly Gly Gly Val Lys Leu Val Lys 645 650 655Glu Val Lys His Ser Arg Leu Leu Leu Arg Glu Cys Leu Arg Ser Asp 660 665 670Ala Gly Glu Ile Lys Ile Gln Leu Lys Asn Pro Phe Gly Thr Ile Glu 675 680 685Ala Thr Ser Arg Leu Ile Val Leu Asp Lys Pro Gly Pro Pro Glu Gly 690 695 700Pro Val Glu Ile Leu Glu Thr Thr Ser Thr Val Ile Glu Leu Gln Trp705 710 715 720Gly Ala Pro Lys Asp Asp Gly Gly Ser Pro Val Thr Asn Tyr Ile Ile 725 730 735Glu Arg Gln Gln Leu Gly Gln Thr Val Trp Lys Lys Met Gly Asp Val 740 745 750Ala Ala Asp Lys Thr Thr Tyr Arg Asp Arg Asn Val Val His Gly Lys 755 760 765Leu Tyr Ile Tyr Asn Ile Tyr Ala Val Asn Pro Glu Gly Thr Ser Asp 770 775 780Ala Leu Gln Thr Glu Glu Thr Met Ala Gly Val Leu Ile Phe Ala Gly785 790 795 800Arg Pro Gly Ala Pro Lys Val Val Ser Ala Ser Lys Thr Cys Ile Asn 805 810 815Leu Lys Trp Glu Pro Pro Glu Asp Asp Gly Gly Ile Lys Ile Asp Gly 820 825 830Tyr Gln Leu Glu Lys Arg Lys Lys Asp Thr Ala Gln Trp Ile Ala Leu 835 840 845Asn Pro Val Thr Glu Pro Ile Glu Val Leu Glu Tyr Ala Val Lys Asp 850 855 860Val Val Glu Gly Ala Glu Tyr Glu Phe Arg Val Ser Ala Ile Asn Val865 870 875 880Ser Gly Ala Gly Glu Phe Ser Leu Pro Ser Val Met Val Thr Ala Lys 885 890 895Asn Pro Asn Met Arg Pro Thr Phe Lys Asp Pro Glu Asp Phe Met Val 900 905 910Ile Arg Ala Gly Asn Ser Val Arg Ile Lys Val Leu Tyr Glu Ala Glu 915 920 925Pro Pro Pro Glu Ile Thr Trp Met Lys Asp Asn Glu Pro Val Ser Ser 930 935 940Phe Ile Gln Ile Ile Asn Thr Glu Gly Cys Ser Gln Leu Val Ile Pro945 950 955 960Ser Thr Lys Arg Ser Asp Ser Gly Asn Tyr Thr Ile Val Ala Lys Asn 965 970 975Lys Val Gly Glu Ala Ser Phe Asp Ile Glu Val Leu Val Thr Asp Glu 980 985 990Pro Lys Pro Pro Gly Ala Val Glu Leu Glu Gln Ile Val His Gly Lys 995 1000 1005Val Ile Val Ser Trp Glu Ala Ser Pro Asp Gln Glu Leu Asp Asn 1010 1015 1020Arg Leu Tyr Tyr Met Val Ala Glu His Asp Ser Ser Thr Arg Met 1025 1030 1035Trp His Thr Val Ala Asp Arg Ile Phe Asp Asn Ser Tyr Thr Ala 1040 1045 1050Asn Asn Ile Met Pro Gly Arg Glu Tyr His Phe Lys Ile Tyr Ala 1055 1060 1065Lys Asn Asp Met Gly Met Ser Asp Pro Ser Met Ser Pro Thr Trp 1070 1075 1080Gly Ile Asn Ser Asn Arg Ile Pro Ile Asn Thr Asn Thr Pro Val 1085 1090 1095Glu Val Ser Phe Glu Lys Pro Pro Ser Val Leu Val Pro Leu Lys 1100 1105 1110Met His Ser Pro Pro Lys Gly Phe Gln Met Tyr Met Thr Cys Ala 1115 1120 1125Ile Arg Gly Cys Pro Thr Pro Ser Val Thr Trp His Leu Asn Asn 1130 1135 1140Val Cys Ile Asn Gly Asp Ser Asn Tyr Tyr Ile Thr Asn Ser Tyr 1145 1150 1155Gly Val Cys Ser Met Tyr Ile Leu Arg Val Arg Pro Lys Asp Ala 1160 1165 1170Gly Glu Tyr Lys Val Val Ala Val Asn Ser Phe Gly Lys Ala Glu 1175 1180 1185Cys Ser Thr Lys Leu Val Val Lys Asp 1190 1195617PRTArtificial SequenceEEF1A2 binding protein-like fragment 6Ala Gln Ala Asp Ser Gln Val Gln Pro Glu Ala Ala Val Ala Asp Phe1 5 10 15Lys710PRTArtificial SequenceEEF1A2 binding protein-like fragment 7Gly Ile Tyr Ala Ala Val Ala Gly Ile Lys1 5 10821PRTArtificial SequenceEEF1A2 binding protein-like fragment 8Thr Thr Arg Ala Gly Gly Ala Gly Val Asp Leu Thr Ala Ile Ala Gln1 5 10 15Glu Gln Ala Val Lys 2092532DNAArtificial SequenceAlpha-1,4 glucan phosphorylase 9atgtcaaaac cgttgtcaga tcacgataga aagaagcaga tctccgtgag aggtcttgct 60ggtgtggaaa atgtggcaga actgaaggtc gctttcaaca ggcatctcca ttttacgctg 120gtcaaggaca gaaatgtggc atccaaacgg gattactact ttgctctcgc caacaccgtg 180cgtgaccact tggtgggcag gtggatcaga acccagcaat actactatga gaaagatccc 240aaacgtgtgt actacatctc cctggagttc tacatgggtc gcaccctgca gaacaccatg 300gtgaacctgg cgctggaaaa cgcctgtgat gaggccatat accagctggg tctggacatg 360gaggagctgg aggacatgga ggaggacgca ggcctgggaa acggtggtct tggacgtctt 420gccgcctgct tcctggactc tatggcttct ctgggtctgg ctgcgtatgg ctacggtatc 480cgctatgagt ttggcatctt taaccagaag atcgtcaatg gatggcaggt tgaggaggct 540gatgactggc tgcgttacgg caacccctgg gagaaggccc gacccgagta catgcgcccc 600gtcaagttct atggcagaac cgagcacacc ccagagggtg tgaaatgggt tgacactcag 660gtagtgttgg ctctgccata tgacacccct atccccgggt acagaaacaa cattgtcaac 720accatgagac tgtggtctgc aaaggcccca tgcgacttca acctgaaaga cttcaacgtt 780ggtgggtaca ttcaggctgt gttggacaga aacctatgcg agaacatttc ccgtgtgctg 840taccccaatg ataacttctt tgagggcaag gagctgcgtc tgaagcagga gtactttgtg 900gtggccgcca cccttcagga catcgtccgt cgtttcaagg cctctaagtt tggctccaga 960gagatcgtcc gcacagactt cgcccagctg cccaacaaag ttgccatcca gctgaatgac 1020actcaccctg ccatggccat tcctgagctg atgagggtac tggttgatga ggagaagctg 1080gagtgggaca aggcctggga cgtgtgtgtc cgtacctgtg cctacacaaa ccacaccgtg 1140ctgcctgagg ccctggagcg ctggcccatt gacctgttcc atcacctgct gccccgtcac 1200ctggagatta tctacgagat caaccgtcgc ttcctgcagt acgtcgcctc gaagttccct 1260ggcgacaacg accgtctgcg ccgcatgtcc ctgattgagg agggagaatg caagaaagtc 1320aacatggctc atatgtgtat cgttggatcc catgctgtca acggcgtggc ccgcatccac 1380tcggagatcc tcgtcgccac tctgttcaag gacttttatg agttggaccc acacaagttc 1440cagaacaaga ccaatggcat caccccccgt cgctggctgg ttatgtgcaa ccccggcttg 1500gctgaggtca tcgcagagag aattggagag gagtttgtcc gtgaccttga ccagctgaag 1560aaactgttga agttcattga tgatgatgct ttcatccgtg acatagccaa agtcaagcag 1620gagaacaagc tgaagttcgc tgtgcacctg gaagaacact acaaagtaaa gatcaacccc 1680cagtccatgt ttgacttcca agtcaaaaga atccacgagt acaagagaca gctgctcaac 1740tgtctgcaca tgatcaccta ctacaaccgt atcaagaagg agccaaacaa gcactggacc 1800ccaagaacca tcatggtcgg aggaaaggct gccccaggct accacacagc caagatgatc 1860attcgtctca tcaccgctat cggtgaggtt gtcaaccacg accccgtgat cggcgaccgc 1920ctcaaagtca tcttcttgga gaactacaga gtcaccctgg ctgagaaagc catcccctct 1980gctgacctgt ctgagcagat ctctacagct ggcactgagg cctctggcac tggcaacatg 2040aagttcatgc tgaatggcgc tctgaccatc ggcaccatgg acggagccaa cgtggagatg 2100gccgaggagg ccggagagaa gaacctcttc atcttcggca tgagagtgga ggaagtggac 2160gcaatggacg ccggcaaagg ataccacgcc tctgagtact acaaccgtat tcccgagctg 2220aaacaggcca tggaccagat ctctggtggc ttcttcagcc ataagcagcc agacctcttc 2280aaggaacttg tggacctgct gatgcaccac gacaggttca aggtgtttgc tgactacgaa 2340gcctacatca aaagtcagga taaggtcaac gaactgtaca agaaacccaa ggaatggacc 2400aagatggtga tccataacat tgcaggctgt ggtaaattct ccagcgaccg caccatttcc 2460cagtacgccc gggagatctg gggcatggag cccagcctgg agaagatccc tgcccccgat 2520gagcaactca aa 253210844PRTArtificial SequenceAlpha-1,4 glucan phosphorylase 10Met Ser Lys Pro Leu Ser Asp His Asp Arg Lys Lys Gln Ile Ser Val1 5 10 15Arg Gly Leu Ala Gly Val Glu Asn Val Ala Glu Leu Lys Val Ala Phe 20 25 30Asn Arg His Leu His Phe Thr Leu Val Lys Asp Arg Asn Val Ala Ser 35 40 45Lys Arg Asp Tyr Tyr Phe Ala Leu Ala Asn Thr Val Arg Asp His Leu 50 55 60Val Gly Arg Trp Ile Arg Thr Gln Gln Tyr Tyr Tyr Glu Lys Asp Pro65 70 75 80Lys Arg Val Tyr Tyr Ile Ser Leu Glu Phe Tyr Met Gly Arg Thr Leu 85 90 95Gln Asn Thr Met Val Asn Leu Ala Leu Glu Asn Ala Cys Asp Glu Ala 100 105 110Ile Tyr Gln Leu Gly Leu Asp Met Glu Glu Leu Glu Asp Met Glu Glu 115 120 125Asp Ala Gly Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ala Cys Phe 130 135 140Leu Asp Ser Met Ala Ser Leu Gly Leu Ala Ala Tyr Gly Tyr Gly Ile145 150 155 160Arg Tyr Glu Phe Gly Ile Phe Asn Gln Lys Ile Val Asn Gly Trp Gln 165 170 175Val Glu Glu Ala Asp Asp Trp Leu Arg Tyr Gly Asn Pro Trp Glu Lys 180 185 190Ala Arg Pro Glu Tyr Met Arg Pro Val Lys Phe Tyr Gly Arg Thr Glu 195 200 205His Thr Pro Glu Gly Val Lys Trp Val Asp Thr Gln Val Val Leu Ala 210 215 220Leu Pro Tyr Asp Thr Pro Ile Pro Gly Tyr Arg Asn Asn Ile Val Asn225 230 235 240Thr Met Arg Leu Trp Ser Ala Lys Ala Pro Cys Asp Phe Asn Leu Lys 245 250 255Asp Phe Asn Val Gly Gly Tyr Ile Gln Ala Val Leu Asp Arg Asn Leu 260 265 270Cys Glu Asn Ile Ser Arg Val Leu Tyr Pro Asn Asp Asn Phe Phe Glu 275 280 285Gly Lys Glu Leu Arg Leu Lys Gln Glu Tyr Phe Val Val Ala Ala Thr 290 295 300Leu Gln Asp Ile Val Arg Arg Phe Lys Ala Ser Lys Phe Gly Ser Arg305 310 315 320Glu Ile Val Arg Thr Asp Phe Ala Gln Leu Pro Asn Lys Val Ala Ile 325 330 335Gln Leu Asn Asp Thr His Pro Ala Met Ala Ile Pro Glu Leu Met Arg 340 345 350Val Leu Val Asp Glu Glu Lys Leu Glu Trp Asp Lys Ala Trp Asp Val 355 360 365Cys Val Arg Thr Cys Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala 370 375 380Leu Glu Arg Trp Pro Ile Asp Leu Phe His His Leu Leu Pro Arg His385 390 395 400Leu Glu Ile Ile Tyr Glu Ile Asn Arg Arg Phe Leu Gln Tyr Val Ala 405 410 415Ser Lys Phe Pro Gly Asp Asn Asp Arg Leu Arg Arg Met Ser Leu Ile 420 425 430Glu Glu Gly Glu Cys Lys Lys Val Asn Met Ala His Met Cys Ile Val 435 440 445Gly Ser His Ala Val Asn Gly Val Ala Arg Ile His Ser Glu Ile Leu 450 455 460Val Ala Thr Leu Phe Lys Asp Phe Tyr Glu Leu Asp Pro His Lys Phe465 470 475 480Gln Asn Lys Thr Asn Gly Ile Thr Pro Arg Arg Trp Leu Val Met Cys 485 490 495Asn Pro Gly Leu Ala Glu Val Ile Ala Glu Arg Ile Gly Glu Glu Phe 500 505 510Val Arg Asp Leu Asp Gln Leu Lys Lys Leu Leu Lys Phe Ile Asp Asp 515 520 525Asp Ala Phe Ile Arg Asp Ile Ala Lys Val Lys Gln Glu Asn Lys Leu 530 535 540Lys Phe Ala Val His Leu Glu Glu His Tyr Lys Val Lys Ile Asn Pro545 550 555 560Gln Ser Met Phe Asp Phe Gln Val Lys Arg Ile His Glu Tyr Lys Arg 565 570 575Gln Leu Leu Asn Cys Leu His Met Ile Thr Tyr Tyr Asn Arg Ile Lys 580 585 590Lys Glu Pro Asn Lys His Trp Thr Pro Arg Thr Ile Met Val Gly Gly 595 600 605Lys Ala Ala Pro Gly Tyr His Thr Ala Lys Met Ile Ile Arg Leu Ile 610 615 620Thr Ala Ile Gly Glu Val Val Asn His Asp Pro Val Ile Gly Asp Arg625 630 635 640Leu Lys Val Ile Phe Leu Glu Asn Tyr Arg Val Thr Leu Ala Glu Lys 645 650 655Ala Ile Pro Ser Ala Asp Leu Ser Glu Gln Ile Ser Thr Ala Gly Thr 660 665 670Glu Ala Ser Gly Thr Gly Asn Met Lys Phe Met Leu Asn Gly Ala Leu 675 680 685Thr Ile Gly Thr Met Asp Gly Ala Asn Val Glu Met Ala Glu Glu Ala 690 695 700Gly Glu Lys Asn Leu Phe Ile Phe Gly Met Arg Val Glu Glu Val Asp705 710 715 720Ala Met Asp Ala Gly Lys Gly Tyr His Ala Ser Glu Tyr Tyr Asn Arg 725 730 735Ile Pro Glu Leu Lys Gln Ala Met Asp Gln Ile Ser Gly Gly Phe Phe 740 745 750Ser His Lys Gln Pro Asp Leu Phe Lys Glu Leu Val Asp Leu Leu Met 755 760 765His His Asp Arg Phe Lys Val Phe Ala Asp Tyr Glu Ala Tyr Ile Lys 770 775 780Ser Gln Asp Lys Val Asn Glu Leu Tyr Lys Lys Pro Lys Glu Trp Thr785 790 795 800Lys Met Val Ile His Asn Ile Ala Gly Cys Gly Lys Phe Ser Ser Asp 805 810 815Arg Thr Ile Ser Gln Tyr Ala Arg Glu Ile Trp Gly Met Glu Pro Ser 820 825 830Leu Glu Lys Ile Pro Ala Pro Asp Glu Gln Leu Lys 835 840119PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 11Ile Pro Ala Pro Asp Glu Gln Leu Lys1 51217PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 12Gln Ile Ser Val Arg Gly Leu Ala Gly Val Glu Asn Val Ala Glu Leu1 5 10 15Lys1325PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 13Ala Ile Pro Ser Ala Asp Leu Ser Glu Gln Ile Ser Thr Ala Gly Thr1 5 10 15Glu Ala Ser Gly Thr Gly Asn Met Lys 20 25149PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 14Asp Phe Tyr Glu Leu Asp Pro His Lys1 51510PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 15Phe Ala Val His Leu Glu Glu His Tyr Lys1 5 101615PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 16Gly Tyr His Ala Ser Glu Tyr Tyr Asn Arg Ile Pro Glu Leu Lys1 5 10 15179PRTArtificial SequenceAlpha-1,4 glucan phosphorylase fragment 17Ile Pro Ala Pro Asp Glu Gln Leu Lys1 5182574DNAArtificial SequenceElongation factor 2 18atggtgaact ttacagtgga ccagatccgt gccatcatgg acaagaaatc caacattcgt 60aacatgtctg tgatcgctca cgtggaccac ggcaagtcca cgctgaccga ctccctggtg 120tctaaagccg ggatcatcgc ggggtctcgc gccggagaga cacgcttcac agacactcgc 180aaagacgagc aggagcgctg tattaccatc aagtccacgg ctatctcgat gtactatgag 240ctgggggaaa acgacatggc cttcatcaag cagtctaagg atgggcttgg cttcctcatc 300aacctgattg actcaccggg ccatgtggac ttctcctctg aggtcacagc cgcccttagg 360gtcaccgacg gcgccctggt ggtggttgac tgcgtctcag gtgtgtgtgt gcagacagag 420accgtgttga ggcaggccat tgctgagcgc atcaagccag tgctgatgat gaacaagatg 480gaccgggccc tgctggagct gcagctggag cctgaggacc tgttccagac cttccagcgc 540atcgtggaga atgtcaacgt catcattgcc acctacggag aggatgaagc gggaccaatg 600ggtgccatca tgattgaccc tgtgattggt accgtggggt ttgggtctgg cctccacggc 660tgggccttca ctctaaagca gtttgctgag atgtacgtga caaagttttc tgccggcaaa 720gacacccagc tgggatcggc ggagaggtgt aagaaggtgg aggacatgat gaagaagctg 780tggggggaga ggttttttga cccagccact gggaagttca gtaagtccaa cctcggccct 840gacggtaaga agctgccccg caccttctct cagctggtcc tggaccctat cttcaaggta 900tttgatgcca tcatgaactt caagaaggat gagacagcca agctgataga gaagctggac 960atcaagctgg actctgagga caaggagaag gagggcaagc ccctgttgaa ggcagtgatg 1020cgtcgctggc tcccagccgg agaagccctg ctccagatga tcaccatcca cctgccctcc 1080cccgtcacgg cccagaagta ccgctgtgag ctgctctacg agggaccagg agacgacgag 1140gccgccatgg gtatcaagaa ctgcgacccc aaggctcccc tgatgatgta catatctaag 1200atggtgccca ccacagacaa gggtcgcttc tatgcctttg gccgtgtgtt ctctggctgt 1260gtgtccaccg gtctgaaggt gcgcatcatg ggaccaaact tcacccctgg gaagaaggaa 1320gacctctaca tcaagcccat ccagaggacc attctgatga tggggcgtta tgtggagccc 1380attgaggatg taccatgtgg gaacatcgtt gggctggttg gagttgacca gtatctgatt 1440aagactggga

ccatcaccac ctttgaacag gcccacaaca tgcgtgtgat gaagttcagc 1500gtcagccctg tggtgagggt ggctgtggag gccaagaacc ctgctgacct gcccaagctg 1560gtggaggggc tgaagcgtct ggccaagtct gaccccatgg tgcagtgtat catcgaggag 1620tctggagagc acatcatcgc cggggccgga gagcttcatc tggagatctg tctcaaggat 1680ctggaggagg accacgccgg cattcccctg aagaaatctg atccagtggt gtcctacagg 1740gagactgtgt ctgaggagtc tgaagtgatg tgtctatcca agtcccctaa caagcacaac 1800cgtctgtaca tgcgggctaa acctttccct gacggcctgg ccgaggacat cgagaagggg 1860gacgtcagcc cccgacagga gctgaaaatc cgcgcccgtt tcctggctga caagtacgag 1920tgggacgtgt cggaggcccg taagatctgg tgcttcggcc ctgacggtac cggtcccaac 1980ctgctgatgg atgttaccaa gggagtccag tacctgaatg agatcaagga cagtgttgtg 2040gctggcttcc agtgggctgt caaggagggt gtgttgtgtg aggagaacat gcgtgcagtc 2100cgcttcgaca tccacgacgt gaccctgcac acagacgcaa ttcaccgcgg tggcggacag 2160atcatcccca cggcccgcag agtgctgtat gcctgccagc tcaccgccca gccacgactc 2220atggagccgg tctacttagt ggagatccag tgcccagagc aggtagtggg tgggatctac 2280ggcgtgctga acaggaagcg aggccatgtg ttcgaggagt cccaggtgat gggcacgccc 2340atgttcatcg tcaaggccta cctgcctgtc aacgagtcat ttgggttcac cgctgacctg 2400cgctccaaca cgggcggcca ggctttcccc cagtgtgtgt ttgatcactg gcagatcctc 2460cagggagatc cccaggaccc caccaccaag accgccattg tggtggccga gaccaggaaa 2520cgcaaggggc tgaaagaggg catcccggcc ctggacaact acctggacaa attg 257419858PRTArtificial SequenceElongation factor 2 19Met Val Asn Phe Thr Val Asp Gln Ile Arg Ala Ile Met Asp Lys Lys1 5 10 15Ser Asn Ile Arg Asn Met Ser Val Ile Ala His Val Asp His Gly Lys 20 25 30Ser Thr Leu Thr Asp Ser Leu Val Ser Lys Ala Gly Ile Ile Ala Gly 35 40 45Ser Arg Ala Gly Glu Thr Arg Phe Thr Asp Thr Arg Lys Asp Glu Gln 50 55 60Glu Arg Cys Ile Thr Ile Lys Ser Thr Ala Ile Ser Met Tyr Tyr Glu65 70 75 80Leu Gly Glu Asn Asp Met Ala Phe Ile Lys Gln Ser Lys Asp Gly Leu 85 90 95Gly Phe Leu Ile Asn Leu Ile Asp Ser Pro Gly His Val Asp Phe Ser 100 105 110Ser Glu Val Thr Ala Ala Leu Arg Val Thr Asp Gly Ala Leu Val Val 115 120 125Val Asp Cys Val Ser Gly Val Cys Val Gln Thr Glu Thr Val Leu Arg 130 135 140Gln Ala Ile Ala Glu Arg Ile Lys Pro Val Leu Met Met Asn Lys Met145 150 155 160Asp Arg Ala Leu Leu Glu Leu Gln Leu Glu Pro Glu Asp Leu Phe Gln 165 170 175Thr Phe Gln Arg Ile Val Glu Asn Val Asn Val Ile Ile Ala Thr Tyr 180 185 190Gly Glu Asp Glu Ala Gly Pro Met Gly Ala Ile Met Ile Asp Pro Val 195 200 205Ile Gly Thr Val Gly Phe Gly Ser Gly Leu His Gly Trp Ala Phe Thr 210 215 220Leu Lys Gln Phe Ala Glu Met Tyr Val Thr Lys Phe Ser Ala Gly Lys225 230 235 240Asp Thr Gln Leu Gly Ser Ala Glu Arg Cys Lys Lys Val Glu Asp Met 245 250 255Met Lys Lys Leu Trp Gly Glu Arg Phe Phe Asp Pro Ala Thr Gly Lys 260 265 270Phe Ser Lys Ser Asn Leu Gly Pro Asp Gly Lys Lys Leu Pro Arg Thr 275 280 285Phe Ser Gln Leu Val Leu Asp Pro Ile Phe Lys Val Phe Asp Ala Ile 290 295 300Met Asn Phe Lys Lys Asp Glu Thr Ala Lys Leu Ile Glu Lys Leu Asp305 310 315 320Ile Lys Leu Asp Ser Glu Asp Lys Glu Lys Glu Gly Lys Pro Leu Leu 325 330 335Lys Ala Val Met Arg Arg Trp Leu Pro Ala Gly Glu Ala Leu Leu Gln 340 345 350Met Ile Thr Ile His Leu Pro Ser Pro Val Thr Ala Gln Lys Tyr Arg 355 360 365Cys Glu Leu Leu Tyr Glu Gly Pro Gly Asp Asp Glu Ala Ala Met Gly 370 375 380Ile Lys Asn Cys Asp Pro Lys Ala Pro Leu Met Met Tyr Ile Ser Lys385 390 395 400Met Val Pro Thr Thr Asp Lys Gly Arg Phe Tyr Ala Phe Gly Arg Val 405 410 415Phe Ser Gly Cys Val Ser Thr Gly Leu Lys Val Arg Ile Met Gly Pro 420 425 430Asn Phe Thr Pro Gly Lys Lys Glu Asp Leu Tyr Ile Lys Pro Ile Gln 435 440 445Arg Thr Ile Leu Met Met Gly Arg Tyr Val Glu Pro Ile Glu Asp Val 450 455 460Pro Cys Gly Asn Ile Val Gly Leu Val Gly Val Asp Gln Tyr Leu Ile465 470 475 480Lys Thr Gly Thr Ile Thr Thr Phe Glu Gln Ala His Asn Met Arg Val 485 490 495Met Lys Phe Ser Val Ser Pro Val Val Arg Val Ala Val Glu Ala Lys 500 505 510Asn Pro Ala Asp Leu Pro Lys Leu Val Glu Gly Leu Lys Arg Leu Ala 515 520 525Lys Ser Asp Pro Met Val Gln Cys Ile Ile Glu Glu Ser Gly Glu His 530 535 540Ile Ile Ala Gly Ala Gly Glu Leu His Leu Glu Ile Cys Leu Lys Asp545 550 555 560Leu Glu Glu Asp His Ala Gly Ile Pro Leu Lys Lys Ser Asp Pro Val 565 570 575Val Ser Tyr Arg Glu Thr Val Ser Glu Glu Ser Glu Val Met Cys Leu 580 585 590Ser Lys Ser Pro Asn Lys His Asn Arg Leu Tyr Met Arg Ala Lys Pro 595 600 605Phe Pro Asp Gly Leu Ala Glu Asp Ile Glu Lys Gly Asp Val Ser Pro 610 615 620Arg Gln Glu Leu Lys Ile Arg Ala Arg Phe Leu Ala Asp Lys Tyr Glu625 630 635 640Trp Asp Val Ser Glu Ala Arg Lys Ile Trp Cys Phe Gly Pro Asp Gly 645 650 655Thr Gly Pro Asn Leu Leu Met Asp Val Thr Lys Gly Val Gln Tyr Leu 660 665 670Asn Glu Ile Lys Asp Ser Val Val Ala Gly Phe Gln Trp Ala Val Lys 675 680 685Glu Gly Val Leu Cys Glu Glu Asn Met Arg Ala Val Arg Phe Asp Ile 690 695 700His Asp Val Thr Leu His Thr Asp Ala Ile His Arg Gly Gly Gly Gln705 710 715 720Ile Ile Pro Thr Ala Arg Arg Val Leu Tyr Ala Cys Gln Leu Thr Ala 725 730 735Gln Pro Arg Leu Met Glu Pro Val Tyr Leu Val Glu Ile Gln Cys Pro 740 745 750Glu Gln Val Val Gly Gly Ile Tyr Gly Val Leu Asn Arg Lys Arg Gly 755 760 765His Val Phe Glu Glu Ser Gln Val Met Gly Thr Pro Met Phe Ile Val 770 775 780Lys Ala Tyr Leu Pro Val Asn Glu Ser Phe Gly Phe Thr Ala Asp Leu785 790 795 800Arg Ser Asn Thr Gly Gly Gln Ala Phe Pro Gln Cys Val Phe Asp His 805 810 815Trp Gln Ile Leu Gln Gly Asp Pro Gln Asp Pro Thr Thr Lys Thr Ala 820 825 830Ile Val Val Ala Glu Thr Arg Lys Arg Lys Gly Leu Lys Glu Gly Ile 835 840 845Pro Ala Leu Asp Asn Tyr Leu Asp Lys Leu 850 8552010PRTArtificial SequenceElongation factor 2 fragment 20Thr Ala Ile Val Val Ala Glu Thr Arg Lys1 5 102112PRTArtificial SequenceElongation factor 2 fragment 21Asp Leu Glu Glu Asp His Ala Gly Ile Pro Leu Lys1 5 102213PRTArtificial SequenceElongation factor 2 fragment 22Asp Leu Glu Glu Asp His Ala Gly Ile Pro Leu Lys Lys1 5 102312PRTArtificial SequenceElongation factor 2 fragment 23Asp Ser Val Val Ala Gly Phe Gln Trp Ala Val Lys1 5 102410PRTArtificial SequenceElongation factor 2 fragment 24Gly Asp Val Ser Pro Arg Gln Glu Leu Lys1 5 10251989DNAArtificial SequenceHeat shock cognate 70 kDa protein 25atgtcaaagg gaccagcagt tggcattgac ctgggcacca cctactcctg tgtgggtgtg 60tttcagcatg gcaaagtgga gatcatagcc aacgaccagg gaaacaggac cacacccagt 120tatgtcgcct tcacagacac agaaaggctg attggggacg cagccaagaa ccaagtggcc 180atgaacccca caaacacagt gtttgatgct aagcggctga tagggcggaa gtttgacgac 240agtgtcgtcc aggcagacat gaaacactgg ccgtttacag tgatcaacga ctcgacacgg 300cccaaggtcc aagtggagta caagggagag accaaggcct tctacccaga ggagatctcc 360tccatggtgc tggtcaagat gaaggagatt gcagaggcct acctgggcaa gactataacc 420aatgcagtgg tcactgtgcc agcttacttc aacgactctc agcgccaggc caccaaagat 480gcagggacta tctcaggact caacgtactc cgcatcatca atgagccaac tgctgctgct 540atcgcctatg gcctggacaa gaaggtggga gtggagagaa acgtcctaat ctttgaccta 600ggcggaggta cgtttgatgt gtctatcctg accatcgaag acgggatctt tgaggtgaag 660tccacggccg gagacaccca tcttggagga gaggacttcg acaaccgcat ggtcaaccac 720ttcatctctg agttcaagcg caaatacaag aaggacatca gcgataacaa gagggccgtg 780cgtcgtctgc gcaccgcctg tgaacgtgcc aagcgcaccc tgtcctccag tacccaggcc 840agcattgaga ttgactctct gtatgagggc gtcgacttct acacctccat caccagggct 900cgctttgagg agctgaacgc tgacctgttc agaggcaccc tggaccccgt ggagaagtct 960ctgagggacg ccaagatgga caaggcccag gttcacgaca tcgtcctagt gggaggctcc 1020acacgcatcc ccaagatcca aaagttgctg caggacttct tcaatgggaa ggagctcaac 1080aagagcatca accctgatga ggcggttgcc tatggtgcag ctgttcaggc ggccatcttg 1140tccggagaca agtctgagaa cgtgcaggac ctgctgctgc tggacgtcac gccactgtcc 1200ctgggcattg agacggccgg aggggtcatg accgtgctca tcaagaggaa caccaccatc 1260cccaccaagc agacgcagac cttcacaaca tactcagaca accagcctgg ggtgctcatt 1320caggtatatg agggggaaag agccatgacc aaagacaaca acctactggg gaagtttgag 1380ttgtgtggga ttccaccagc cccccggggt gtgcctcaga tcgaggtcac ctttgacatt 1440gacgccaacg gcatcatgaa cgtgtctgcc gctgacaaga gcaccggcaa ggagaataag 1500atcaccatca ccaatgacaa aggtcgtctg agtaaggagg acatagagcg catggtccag 1560gaggctgaac agtacaaagc tgcggacgat gtccagaggg acaaggtggc gtccaagaat 1620ggcctggagt catacgcctt caacatgaag tccaccgtgg aggacgagaa gctcaagggc 1680aagctcagcg acgaggacaa acagaagatc ctggacaaat gcaatgaggt catcagctgg 1740ctggacaaga accagtctgc agagaaggag gagtttgagc accaccagaa ggagctggag 1800aaggtctgta accccatcat caccaagctg taccagggtg ctggggggat gcctggaggg 1860atgcctggag ggatgcctgg agggatgcct ggagggatgc ctggggggat gcctggaggg 1920atgcccgggg ctggtggtgc cgcacccgga ggaggtggat cctctggacc aacaattgag 1980gaagtcgac 198926663PRTArtificial SequenceHeat shock cognate 70 kDa protein 26Met Ser Lys Gly Pro Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser1 5 10 15Cys Val Gly Val Phe Gln His Gly Lys Val Glu Ile Ile Ala Asn Asp 20 25 30Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu 35 40 45Arg Leu Ile Gly Asp Ala Ala Lys Asn Gln Val Ala Met Asn Pro Thr 50 55 60Asn Thr Val Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Asp Asp65 70 75 80Ser Val Val Gln Ala Asp Met Lys His Trp Pro Phe Thr Val Ile Asn 85 90 95Asp Ser Thr Arg Pro Lys Val Gln Val Glu Tyr Lys Gly Glu Thr Lys 100 105 110Ala Phe Tyr Pro Glu Glu Ile Ser Ser Met Val Leu Val Lys Met Lys 115 120 125Glu Ile Ala Glu Ala Tyr Leu Gly Lys Thr Ile Thr Asn Ala Val Val 130 135 140Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys Asp145 150 155 160Ala Gly Thr Ile Ser Gly Leu Asn Val Leu Arg Ile Ile Asn Glu Pro 165 170 175Thr Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Val Gly Val Glu 180 185 190Arg Asn Val Leu Ile Phe Asp Leu Gly Gly Gly Thr Phe Asp Val Ser 195 200 205Ile Leu Thr Ile Glu Asp Gly Ile Phe Glu Val Lys Ser Thr Ala Gly 210 215 220Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met Val Asn His225 230 235 240Phe Ile Ser Glu Phe Lys Arg Lys Tyr Lys Lys Asp Ile Ser Asp Asn 245 250 255Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg 260 265 270Thr Leu Ser Ser Ser Thr Gln Ala Ser Ile Glu Ile Asp Ser Leu Tyr 275 280 285Glu Gly Val Asp Phe Tyr Thr Ser Ile Thr Arg Ala Arg Phe Glu Glu 290 295 300Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu Asp Pro Val Glu Lys Ser305 310 315 320Leu Arg Asp Ala Lys Met Asp Lys Ala Gln Val His Asp Ile Val Leu 325 330 335Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Lys Leu Leu Gln Asp 340 345 350Phe Phe Asn Gly Lys Glu Leu Asn Lys Ser Ile Asn Pro Asp Glu Ala 355 360 365Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Ser Gly Asp Lys 370 375 380Ser Glu Asn Val Gln Asp Leu Leu Leu Leu Asp Val Thr Pro Leu Ser385 390 395 400Leu Gly Ile Glu Thr Ala Gly Gly Val Met Thr Val Leu Ile Lys Arg 405 410 415Asn Thr Thr Ile Pro Thr Lys Gln Thr Gln Thr Phe Thr Thr Tyr Ser 420 425 430Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr Glu Gly Glu Arg Ala 435 440 445Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe Glu Leu Cys Gly Ile 450 455 460Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe Asp Ile465 470 475 480Asp Ala Asn Gly Ile Met Asn Val Ser Ala Ala Asp Lys Ser Thr Gly 485 490 495Lys Glu Asn Lys Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu Ser Lys 500 505 510Glu Asp Ile Glu Arg Met Val Gln Glu Ala Glu Gln Tyr Lys Ala Ala 515 520 525Asp Asp Val Gln Arg Asp Lys Val Ala Ser Lys Asn Gly Leu Glu Ser 530 535 540Tyr Ala Phe Asn Met Lys Ser Thr Val Glu Asp Glu Lys Leu Lys Gly545 550 555 560Lys Leu Ser Asp Glu Asp Lys Gln Lys Ile Leu Asp Lys Cys Asn Glu 565 570 575Val Ile Ser Trp Leu Asp Lys Asn Gln Ser Ala Glu Lys Glu Glu Phe 580 585 590Glu His His Gln Lys Glu Leu Glu Lys Val Cys Asn Pro Ile Ile Thr 595 600 605Lys Leu Tyr Gln Gly Ala Gly Gly Met Pro Gly Gly Met Pro Gly Gly 610 615 620Met Pro Gly Gly Met Pro Gly Gly Met Pro Gly Gly Met Pro Gly Gly625 630 635 640Met Pro Gly Ala Gly Gly Ala Ala Pro Gly Gly Gly Gly Ser Ser Gly 645 650 655Pro Thr Ile Glu Glu Val Asp 6602714PRTArtificial SequenceHeat shock cognate 70 kDa protein fragment 27Ala Phe Tyr Pro Glu Glu Ile Ser Ser Met Val Leu Val Lys1 5 102814PRTArtificial SequenceHeat shock cognate 70 kDa protein fragment 28Glu Asp Ile Glu Arg Met Val Gln Glu Ala Glu Gln Tyr Lys1 5 102914PRTArtificial SequenceHeat shock cognate 70 kDa protein fragment 29His Trp Pro Phe Thr Val Ile Asn Asp Ser Thr Arg Pro Lys1 5 103014PRTArtificial SequenceHeat shock cognate 70 kDa protein fragment 30Asn Gln Ser Ala Glu Lys Glu Glu Phe Glu His His Gln Lys1 5 103122PRTArtificial SequenceHeat shock cognate 70 kDa protein fragment 31Thr Ile Thr Asn Ala Val Val Thr Val Pro Ala Tyr Phe Asn Asp Ser1 5 10 15Gln Arg Gln Ala Thr Lys 20322061DNAArtificial SequenceSerotransferrin 32atgaaactgc ttctcgtctc agcgctgctg gggtgcttcg ctacggtgta tgctgcccca 60gctgagggaa tggttagatg gtgcgtaaag tcggaaaaag agctgaagaa atgccacgat 120ctcgcagcca atgtggcggg gttttcatgc gtgaggaggg acgactctct tgaatgcatc 180caagccatca agagggaaga ggctgatgct atcactctgg atggaggaga tatttacata 240gctggcctcc acaactacaa cctgcagccc atcattgcag aggactatgg tgaggactct 300gacacctgct attatgctgt ggccgtggcc aaaaagggca ctgaatttgg gttcctcgac 360ctccgtggca agaagtcctg ccacaccggg ttgggcaaat ctgcaggctg gaacattccc 420atcggtaccc tggtgactgt gggccagatc caatgggccg gcatcgagga cagacctgtg 480gagtcggcgg tgagcgactt cttcaatgcc agctgtgctc caggagccaa tagggattct 540caactgtgcc agttgtgtat gggagactgc tccagatctc acaacgagcc ctactatgac 600tattccgggg ccttccagtg cctgaaggat ggagcaggag aggttgcctt catcaagcac 660ctgactgtac ctgccgcaga gaaggcaagc tatgagttgc tgtgcaagga taacaccaga 720gctcccatcg acagctacaa gacctgccac ctggccagag tacccgccca cgctgtggtc 780agccgcaagg accccaggct ggccaatctc atctacagca agctgatggc cgtcacgaac 840ttcaacctgt tctcctccga tggttatgct gccaagaacc tgatgttcaa ggactccact 900cagaatctag tacagctgcc aatgaccacc gactccttcc tctacctggg agctgagtac 960atgagcacta tacgctccct gacaaaagcg caggccacag gtgtcacctc cagggccatc 1020aaatggtgtg ccgtgggcca taaggagaag gtcaagtgtg

acgcctggac aatcaacagc 1080ttcacagatg gtgactccag gatcgaatgc caggacgcac ccacagtgga tgaatgcatc 1140aagaagatca tgcgtaaaga ggcagatgcc atagcggtgg atggtgggga ggtgttcact 1200gctggaaaat gtggtctggt ccctgtcatg gtggagcagt atgatgaagt tcggtgcagc 1260gcccctggtg aggcgtcatc ctactttgcg gtggcggtgg caaagagggg atctgggttg 1320acctggacaa ccctgaaggg caagaggtca tgccacaccg gcttgggcag gaccgcaggc 1380tggaacatac ccatgggtct tatccatagg aggactatga actgcgactt caccacatac 1440ttcagtaagg gctgtgctcc tggatttgag gtggactctc ccttctgtgc ccagtgtaag 1500ggcagtggga agtccgtggg aggagatggg tccaagtgca aagccagctc tgaagagcag 1560tactacggct ataacggagc attcagatgt ctggttgaag atgctggaga tgttgccttc 1620attaaacaca ctattgtacc agagatgact gatggtagtg gtccagtttg ggcacagaac 1680ctgatgtcct ctgactttga actactctgc caggatggta ctaccaagcc agtcacacat 1740ttccgtgagt gccacctggc caaggtgccc gcccatgctg tgataacacg cccagagtcc 1800cgtggagagg ttgtgtccat ccttctggag cagcaggcca ggttcggctc aagcggcagc 1860gattcctcat ttaatatgtt caagccagat tttggaaaga acttgctctt caaggattcc 1920acaaagtgtc tccaggagat cccaagcggc accaaattcc agggtttcct tggggaagag 1980tacatgatcg ccatgcaatc gctcagggag tgctccaaca gtacctcaga tttggagaag 2040gcatgcactt tccattcctg c 206133687PRTArtificial SequenceSerotransferrin 33Met Lys Leu Leu Leu Val Ser Ala Leu Leu Gly Cys Phe Ala Thr Val1 5 10 15Tyr Ala Ala Pro Ala Glu Gly Met Val Arg Trp Cys Val Lys Ser Glu 20 25 30Lys Glu Leu Lys Lys Cys His Asp Leu Ala Ala Asn Val Ala Gly Phe 35 40 45Ser Cys Val Arg Arg Asp Asp Ser Leu Glu Cys Ile Gln Ala Ile Lys 50 55 60Arg Glu Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Asp Ile Tyr Ile65 70 75 80Ala Gly Leu His Asn Tyr Asn Leu Gln Pro Ile Ile Ala Glu Asp Tyr 85 90 95Gly Glu Asp Ser Asp Thr Cys Tyr Tyr Ala Val Ala Val Ala Lys Lys 100 105 110Gly Thr Glu Phe Gly Phe Leu Asp Leu Arg Gly Lys Lys Ser Cys His 115 120 125Thr Gly Leu Gly Lys Ser Ala Gly Trp Asn Ile Pro Ile Gly Thr Leu 130 135 140Val Thr Val Gly Gln Ile Gln Trp Ala Gly Ile Glu Asp Arg Pro Val145 150 155 160Glu Ser Ala Val Ser Asp Phe Phe Asn Ala Ser Cys Ala Pro Gly Ala 165 170 175Asn Arg Asp Ser Gln Leu Cys Gln Leu Cys Met Gly Asp Cys Ser Arg 180 185 190Ser His Asn Glu Pro Tyr Tyr Asp Tyr Ser Gly Ala Phe Gln Cys Leu 195 200 205Lys Asp Gly Ala Gly Glu Val Ala Phe Ile Lys His Leu Thr Val Pro 210 215 220Ala Ala Glu Lys Ala Ser Tyr Glu Leu Leu Cys Lys Asp Asn Thr Arg225 230 235 240Ala Pro Ile Asp Ser Tyr Lys Thr Cys His Leu Ala Arg Val Pro Ala 245 250 255His Ala Val Val Ser Arg Lys Asp Pro Arg Leu Ala Asn Leu Ile Tyr 260 265 270Ser Lys Leu Met Ala Val Thr Asn Phe Asn Leu Phe Ser Ser Asp Gly 275 280 285Tyr Ala Ala Lys Asn Leu Met Phe Lys Asp Ser Thr Gln Asn Leu Val 290 295 300Gln Leu Pro Met Thr Thr Asp Ser Phe Leu Tyr Leu Gly Ala Glu Tyr305 310 315 320Met Ser Thr Ile Arg Ser Leu Thr Lys Ala Gln Ala Thr Gly Val Thr 325 330 335Ser Arg Ala Ile Lys Trp Cys Ala Val Gly His Lys Glu Lys Val Lys 340 345 350Cys Asp Ala Trp Thr Ile Asn Ser Phe Thr Asp Gly Asp Ser Arg Ile 355 360 365Glu Cys Gln Asp Ala Pro Thr Val Asp Glu Cys Ile Lys Lys Ile Met 370 375 380Arg Lys Glu Ala Asp Ala Ile Ala Val Asp Gly Gly Glu Val Phe Thr385 390 395 400Ala Gly Lys Cys Gly Leu Val Pro Val Met Val Glu Gln Tyr Asp Glu 405 410 415Val Arg Cys Ser Ala Pro Gly Glu Ala Ser Ser Tyr Phe Ala Val Ala 420 425 430Val Ala Lys Arg Gly Ser Gly Leu Thr Trp Thr Thr Leu Lys Gly Lys 435 440 445Arg Ser Cys His Thr Gly Leu Gly Arg Thr Ala Gly Trp Asn Ile Pro 450 455 460Met Gly Leu Ile His Arg Arg Thr Met Asn Cys Asp Phe Thr Thr Tyr465 470 475 480Phe Ser Lys Gly Cys Ala Pro Gly Phe Glu Val Asp Ser Pro Phe Cys 485 490 495Ala Gln Cys Lys Gly Ser Gly Lys Ser Val Gly Gly Asp Gly Ser Lys 500 505 510Cys Lys Ala Ser Ser Glu Glu Gln Tyr Tyr Gly Tyr Asn Gly Ala Phe 515 520 525Arg Cys Leu Val Glu Asp Ala Gly Asp Val Ala Phe Ile Lys His Thr 530 535 540Ile Val Pro Glu Met Thr Asp Gly Ser Gly Pro Val Trp Ala Gln Asn545 550 555 560Leu Met Ser Ser Asp Phe Glu Leu Leu Cys Gln Asp Gly Thr Thr Lys 565 570 575Pro Val Thr His Phe Arg Glu Cys His Leu Ala Lys Val Pro Ala His 580 585 590Ala Val Ile Thr Arg Pro Glu Ser Arg Gly Glu Val Val Ser Ile Leu 595 600 605Leu Glu Gln Gln Ala Arg Phe Gly Ser Ser Gly Ser Asp Ser Ser Phe 610 615 620Asn Met Phe Lys Pro Asp Phe Gly Lys Asn Leu Leu Phe Lys Asp Ser625 630 635 640Thr Lys Cys Leu Gln Glu Ile Pro Ser Gly Thr Lys Phe Gln Gly Phe 645 650 655Leu Gly Glu Glu Tyr Met Ile Ala Met Gln Ser Leu Arg Glu Cys Ser 660 665 670Asn Ser Thr Ser Asp Leu Glu Lys Ala Cys Thr Phe His Ser Cys 675 680 6853412PRTArtificial SequenceSerotransferrin fragment 34Ala Gln Ala Thr Gly Val Thr Ser Arg Ala Ile Lys1 5 103510PRTArtificial SequenceSerotransferrin fragment 35Ala Thr Gly Val Thr Ser Arg Ala Ile Lys1 5 103611PRTArtificial SequenceSerotransferrin fragment 36Asp Pro Arg Leu Ala Asn Leu Ile Tyr Ser Lys1 5 103712PRTArtificial SequenceSerotransferrin fragment 37Gly Thr Glu Phe Gly Phe Leu Asp Leu Arg Gly Lys1 5 103813PRTArtificial SequenceSerotransferrin fragment 38Lys Gly Thr Glu Phe Gly Phe Leu Asp Leu Arg Gly Lys1 5 103914PRTArtificial SequenceSerotransferrin fragment 39Lys Gly Thr Glu Phe Gly Phe Leu Asp Leu Arg Gly Lys Lys1 5 104018PRTArtificial SequenceSerotransferrin fragment 40Leu Met Ala Val Thr Asn Phe Asn Leu Phe Ser Ser Asp Gly Tyr Ala1 5 10 15Ala Lys4111PRTArtificial SequenceSerotransferrin fragment 41Arg Gly Ser Gly Leu Thr Trp Thr Thr Leu Lys1 5 10421518DNAArtificial SequenceMyosin binding protein H-like 42atgcctgcga agcctgcccc tatcaagaag gcagccccga aagagcctgc tcctaagaag 60gaggaagcac ctgcacctgc acccgcggag gcggctcctg ctgaagttgc tccccctgcc 120gaggtcgaag ctccaccggc agaggctgcc cctgccgaag cagctgcccc tgctgaggca 180gctccaaccg aagaaaaggc tcccgcagaa cccgcgcctg agcagcccaa agcacccaca 240ccccctcctc cagatgagcc caccagtgag cccctggaag tgacagtgga cgacaagagc 300gacacttcag tgaccatcac ctggaggcct ccaaagacta ttccagccaa ctctggcctg 360gatggataca ctgtggaaat caccaaggag ggaactgaag actggaaagc agccaacgag 420gatctgaccg tgtcctgccg ttatgtcatc aagaaccaga ctaccggcga ccgtctgctg 480atccgagtgg ttgctgtaaa ccctggtggc cgcagccccc ctgctaccat cgccgacccc 540gttctggtca aggaggttgg ggctcgcccc aaagtccgcc tccctcgttt cctgaggcag 600aggtatgtca agaaagtcgg cgagaagatc aacattgtca tcccgtactc tggcaaacct 660aagccagtgg ttagctggtt aaaggatgga cagcccctcg actcaaagag ggccaacatc 720cgtacatctg acagagacag catcctgttc atccgtcaag cagagagggt ggactctggc 780tcatacgaaa tgtgcgtgaa ggtggacgac tttgaggaca aagctgctat catcatccag 840attattgagt tacccgggcc accagccagc attaagattg tggatgtctg gggattcaac 900gttgctttgg agtggaccgt acccaaagat aatggaaaca cagagatcac aggctacact 960gtccagaaag ctgacaagaa gaccggggac tggttcaaca ttttggagca ctacgccaga 1020ctaaatgcca ccatctcaga cctcattatg ggcaacacct acaccttcag ggtctttgca 1080gagaacaagt gcggactcag tgaggaatgt gccgtgacca agggagaagc caccattgtg 1140aaggaggtta ttgactacaa acctaccccg ttcgtggagc atgacttcac cgaggctccc 1200aagttcacca ctgttctgaa cgacaggtcc accactgttg gctacagcac caagctgctg 1260tgttctgtga ggggatgccc caagcctaag attatgtgga tgaagaacaa gatgattctc 1320aacaacatgg acaaccccaa gtacaggatg atcagcacag ggggcatctg caccctggag 1380atccgtaagc ctggcccata cgacggtggt gagtatgtct gcagagccga gaatacactg 1440gggaaggtcg acaccggctg caagctggag gtcagaaagc ctgggcaagc agatgctgac 1500aaagacaaga aggaacag 151843506PRTArtificial SequenceMyosin binding protein H-like 43Met Pro Ala Lys Pro Ala Pro Ile Lys Lys Ala Ala Pro Lys Glu Pro1 5 10 15Ala Pro Lys Lys Glu Glu Ala Pro Ala Pro Ala Pro Ala Glu Ala Ala 20 25 30Pro Ala Glu Val Ala Pro Pro Ala Glu Val Glu Ala Pro Pro Ala Glu 35 40 45Ala Ala Pro Ala Glu Ala Ala Ala Pro Ala Glu Ala Ala Pro Thr Glu 50 55 60Glu Lys Ala Pro Ala Glu Pro Ala Pro Glu Gln Pro Lys Ala Pro Thr65 70 75 80Pro Pro Pro Pro Asp Glu Pro Thr Ser Glu Pro Leu Glu Val Thr Val 85 90 95Asp Asp Lys Ser Asp Thr Ser Val Thr Ile Thr Trp Arg Pro Pro Lys 100 105 110Thr Ile Pro Ala Asn Ser Gly Leu Asp Gly Tyr Thr Val Glu Ile Thr 115 120 125Lys Glu Gly Thr Glu Asp Trp Lys Ala Ala Asn Glu Asp Leu Thr Val 130 135 140Ser Cys Arg Tyr Val Ile Lys Asn Gln Thr Thr Gly Asp Arg Leu Leu145 150 155 160Ile Arg Val Val Ala Val Asn Pro Gly Gly Arg Ser Pro Pro Ala Thr 165 170 175Ile Ala Asp Pro Val Leu Val Lys Glu Val Gly Ala Arg Pro Lys Val 180 185 190Arg Leu Pro Arg Phe Leu Arg Gln Arg Tyr Val Lys Lys Val Gly Glu 195 200 205Lys Ile Asn Ile Val Ile Pro Tyr Ser Gly Lys Pro Lys Pro Val Val 210 215 220Ser Trp Leu Lys Asp Gly Gln Pro Leu Asp Ser Lys Arg Ala Asn Ile225 230 235 240Arg Thr Ser Asp Arg Asp Ser Ile Leu Phe Ile Arg Gln Ala Glu Arg 245 250 255Val Asp Ser Gly Ser Tyr Glu Met Cys Val Lys Val Asp Asp Phe Glu 260 265 270Asp Lys Ala Ala Ile Ile Ile Gln Ile Ile Glu Leu Pro Gly Pro Pro 275 280 285Ala Ser Ile Lys Ile Val Asp Val Trp Gly Phe Asn Val Ala Leu Glu 290 295 300Trp Thr Val Pro Lys Asp Asn Gly Asn Thr Glu Ile Thr Gly Tyr Thr305 310 315 320Val Gln Lys Ala Asp Lys Lys Thr Gly Asp Trp Phe Asn Ile Leu Glu 325 330 335His Tyr Ala Arg Leu Asn Ala Thr Ile Ser Asp Leu Ile Met Gly Asn 340 345 350Thr Tyr Thr Phe Arg Val Phe Ala Glu Asn Lys Cys Gly Leu Ser Glu 355 360 365Glu Cys Ala Val Thr Lys Gly Glu Ala Thr Ile Val Lys Glu Val Ile 370 375 380Asp Tyr Lys Pro Thr Pro Phe Val Glu His Asp Phe Thr Glu Ala Pro385 390 395 400Lys Phe Thr Thr Val Leu Asn Asp Arg Ser Thr Thr Val Gly Tyr Ser 405 410 415Thr Lys Leu Leu Cys Ser Val Arg Gly Cys Pro Lys Pro Lys Ile Met 420 425 430Trp Met Lys Asn Lys Met Ile Leu Asn Asn Met Asp Asn Pro Lys Tyr 435 440 445Arg Met Ile Ser Thr Gly Gly Ile Cys Thr Leu Glu Ile Arg Lys Pro 450 455 460Gly Pro Tyr Asp Gly Gly Glu Tyr Val Cys Arg Ala Glu Asn Thr Leu465 470 475 480Gly Lys Val Asp Thr Gly Cys Lys Leu Glu Val Arg Lys Pro Gly Gln 485 490 495Ala Asp Ala Asp Lys Asp Lys Lys Glu Gln 500 5054418PRTArtificial SequenceMyosin binding protein H-like fragment 44Ala Ala Ile Ile Ile Gln Ile Ile Glu Leu Pro Gly Pro Pro Ala Ser1 5 10 15Ile Lys4518PRTArtificial SequenceMyosin binding protein H-like fragment 45Ala Ala Pro Ala Glu Ala Ala Ala Pro Ala Glu Ala Ala Pro Thr Glu1 5 10 15Glu Lys4611PRTArtificial SequenceMyosin binding protein H-like fragment 46Ala Pro Ala Glu Pro Ala Pro Glu Gln Pro Lys1 5 104714PRTArtificial SequenceMyosin binding protein H-like fragment 47Asp Asn Gly Asn Thr Glu Ile Thr Gly Tyr Thr Val Gln Lys1 5 104820PRTArtificial SequenceMyosin binding protein H-like fragment 48Glu Val Ile Asp Tyr Lys Pro Thr Pro Phe Val Glu His Asp Phe Thr1 5 10 15Glu Ala Pro Lys 204917PRTArtificial SequenceMyosin binding protein H-like fragment 49Phe Thr Thr Val Leu Asn Asp Arg Ser Thr Thr Val Gly Tyr Ser Thr1 5 10 15Lys5010PRTArtificial SequenceMyosin binding protein H-like fragment 50Ile Asn Ile Val Ile Pro Tyr Ser Gly Lys1 5 105119PRTArtificial SequenceMyosin binding protein H-like fragment 51Ile Asn Ile Val Ile Pro Tyr Ser Gly Lys Pro Lys Pro Val Val Ser1 5 10 15Trp Leu Lys5233PRTArtificial SequenceMyosin binding protein H-like fragment 52Asn Gln Thr Thr Gly Asp Arg Leu Leu Ile Arg Val Val Ala Val Asn1 5 10 15Pro Gly Gly Arg Ser Pro Pro Ala Thr Ile Ala Asp Pro Val Leu Val 20 25 30Lys5313PRTArtificial SequenceMyosin binding protein H-like fragment 53Ser Asp Thr Ser Val Thr Ile Thr Trp Arg Pro Pro Lys1 5 105417PRTArtificial SequenceMyosin binding protein H-like fragment 54Thr Ile Pro Ala Asn Ser Gly Leu Asp Gly Tyr Thr Val Glu Ile Thr1 5 10 15Lys551377DNAArtificial SequenceDesmin (fragment) 55atttcctctt accgccgcac tttcggttct ggtattggat ccaccccagg catgtcctcc 60atgttctccc acggtggacg cggctcctcc ggctcggccc acatgtcctc cagagtctac 120gagatgacca aaagctccgc ccgccccagc tactcctccg gcagtattcg ctcctcctcc 180ggcggcgcga tgcgctcgta cgccgggatg ggcgagaagc tggacttcaa cctggccgac 240gccactaacc gcgacttcct ggacaccaga accaatgaga aggcagagtt gcagcacctg 300aacgaccggt tcgccagcta catcgagaag gtccgtttcc tggagcagca gaacgctact 360ctggtggtgg agatcgagag gctgaggggt cacgagccga cccgcgtggc cgagatgtac 420gaggaggaga tgagagagct gaggcgtcag gtggacggca tgtccaatga ccgagcccgc 480atggaggtgg agagagacaa cttggctgac gacctgcaga aactcaaact cagactgcag 540gaggtgatcc accagaggga agaggcagag aacaacctgt ctgccttcag agctgacgtg 600gactctgcca cgctggccag gctggacctg gagagacgca ttgaaagcct gcaggaggag 660atcaccttcc tcaagaagat ccacgaggag gagatccatg agctgacgag ccagatgcag 720gagacctcgg tgcaggtcca gatggacatg tccaaaccag acctgaccgt tgccctcagg 780gacatccgta tgcagtacga gggtatcgca gccaagaaca tctctgaggc agaggactgg 840tacaagtcca aggtgtcaga cctgaaccag gctgtgaaca agaacaacga tgctctgaga 900caggccaaac aggagagcat ggagttcagg catcagatcc agtcctacac ctgtgagatc 960gactctctca agggaaccaa cgagtctctg ctgaggcaga tgagggagat ggaggatcgt 1020ctgggaaacg aggctggagg ttaccaggac tccgtcaccc gtctggaggc tgagatcgcc 1080aagatgaagg atgagatggc tcgtcacctc agagagtacc aggacctcct caatgtcaag 1140atggccctgg atatagagat cgctacctac aggaagctac tggagggaga ggagagccga 1200atcactgtat ctggctccaa gtcttctcac tctggctccc actctgctgc ctcattatat 1260tccactgttg gattcagaga gaccagccca gacgtcggac gatcagcaga ggtccactcc 1320aagaagactg tcctgatcaa gaccatcgag acccgcgatg gagaggtcgt cagcgag 137756459PRTArtificial SequenceDesmin (fragment) 56Ile Ser Ser Tyr Arg Arg Thr Phe Gly Ser Gly Ile Gly Ser Thr Pro1 5 10 15Gly Met Ser Ser Met Phe Ser His Gly Gly Arg Gly Ser Ser Gly Ser 20 25 30Ala His Met Ser Ser Arg Val Tyr Glu Met Thr Lys Ser Ser Ala Arg 35 40 45Pro Ser Tyr Ser Ser Gly Ser Ile Arg Ser Ser Ser Gly Gly Ala Met 50 55 60Arg Ser Tyr Ala Gly Met Gly Glu Lys Leu Asp Phe Asn Leu Ala Asp65 70 75 80Ala Thr Asn Arg Asp Phe Leu Asp Thr Arg Thr Asn Glu Lys Ala Glu 85 90 95Leu Gln His Leu Asn Asp Arg Phe Ala Ser Tyr Ile Glu Lys Val Arg 100 105 110Phe Leu Glu Gln Gln Asn Ala Thr Leu Val Val Glu Ile Glu Arg Leu 115 120 125Arg Gly His Glu Pro Thr Arg

Val Ala Glu Met Tyr Glu Glu Glu Met 130 135 140Arg Glu Leu Arg Arg Gln Val Asp Gly Met Ser Asn Asp Arg Ala Arg145 150 155 160Met Glu Val Glu Arg Asp Asn Leu Ala Asp Asp Leu Gln Lys Leu Lys 165 170 175Leu Arg Leu Gln Glu Val Ile His Gln Arg Glu Glu Ala Glu Asn Asn 180 185 190Leu Ser Ala Phe Arg Ala Asp Val Asp Ser Ala Thr Leu Ala Arg Leu 195 200 205Asp Leu Glu Arg Arg Ile Glu Ser Leu Gln Glu Glu Ile Thr Phe Leu 210 215 220Lys Lys Ile His Glu Glu Glu Ile His Glu Leu Thr Ser Gln Met Gln225 230 235 240Glu Thr Ser Val Gln Val Gln Met Asp Met Ser Lys Pro Asp Leu Thr 245 250 255Val Ala Leu Arg Asp Ile Arg Met Gln Tyr Glu Gly Ile Ala Ala Lys 260 265 270Asn Ile Ser Glu Ala Glu Asp Trp Tyr Lys Ser Lys Val Ser Asp Leu 275 280 285Asn Gln Ala Val Asn Lys Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln 290 295 300Glu Ser Met Glu Phe Arg His Gln Ile Gln Ser Tyr Thr Cys Glu Ile305 310 315 320Asp Ser Leu Lys Gly Thr Asn Glu Ser Leu Leu Arg Gln Met Arg Glu 325 330 335Met Glu Asp Arg Leu Gly Asn Glu Ala Gly Gly Tyr Gln Asp Ser Val 340 345 350Thr Arg Leu Glu Ala Glu Ile Ala Lys Met Lys Asp Glu Met Ala Arg 355 360 365His Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val Lys Met Ala Leu Asp 370 375 380Ile Glu Ile Ala Thr Tyr Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg385 390 395 400Ile Thr Val Ser Gly Ser Lys Ser Ser His Ser Gly Ser His Ser Ala 405 410 415Ala Ser Leu Tyr Ser Thr Val Gly Phe Arg Glu Thr Ser Pro Asp Val 420 425 430Gly Arg Ser Ala Glu Val His Ser Lys Lys Thr Val Leu Ile Lys Thr 435 440 445Ile Glu Thr Arg Asp Gly Glu Val Val Ser Glu 450 4555717PRTArtificial SequenceA part of Desmin (fragment) 57Asp Glu Met Ala Arg His Leu Arg Glu Tyr Gln Asp Leu Leu Asn Val1 5 10 15Lys5821PRTArtificial SequenceA part of Desmin (fragment) 58Leu Asp Phe Asn Leu Ala Asp Ala Thr Asn Arg Asp Phe Leu Asp Thr1 5 10 15Arg Thr Asn Glu Lys 205910PRTArtificial SequenceA part of Desmin (fragment) 59Asn Ile Ser Glu Ala Glu Asp Trp Tyr Lys1 5 1060822DNAArtificial SequenceCapping protein (Actin filament) muscle Z-line beta 60atgaatgacc agcagctgga ctgtgctctg gacctgatga ggcgtcttcc tccccagcag 60atcgagaaga atctcagtga cctcatcgac ctggtgccca gtctgtgtga ggacctactc 120tcctctgtgg accagcctct gaagatcgcc cgggacaagg tagtggggaa agactacctg 180ctctgtgact ataacagaga cggtgactcc tacagatccc catggagtaa taagtatgag 240ccacccatcg acgatggtgc catgccatct gcccgcctgc gcaaactaga ggtggaagcc 300aacaatgcct ttgaccagta tagagacctg tactttgagg gtggcgtatc gtctgtgtat 360ctgtgggact tggatcatgg ctttgctggg gtcatcctca tcaagaaggc tggagacggc 420tccaagaaga tcaaaggctg ctgggactcc atccatgtgg tggaggtgca ggagaagtcc 480agcggacgga ccgctcacta caaactcacc tccaccgtca tgctgtggct ccagacgacc 540aaggccgggt ctggaaccat gaacctgggt ggcagtctga caagacagat ggagaaagac 600gagacagttg gagagtcttc cccacatatt gccaacatcg gccgcctggt ggaggatatg 660gagaataaga ttcgctccac tctcaacgag atctactttg ggaagaccaa ggacatcgtc 720aatggtttaa gatctattga ctctctgcct gataaccaaa agtaccggca gctccagaag 780gagctgtctc aggtccttac ccagcgccag atcttcattg ac 82261274PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta 61Met Asn Asp Gln Gln Leu Asp Cys Ala Leu Asp Leu Met Arg Arg Leu1 5 10 15Pro Pro Gln Gln Ile Glu Lys Asn Leu Ser Asp Leu Ile Asp Leu Val 20 25 30Pro Ser Leu Cys Glu Asp Leu Leu Ser Ser Val Asp Gln Pro Leu Lys 35 40 45Ile Ala Arg Asp Lys Val Val Gly Lys Asp Tyr Leu Leu Cys Asp Tyr 50 55 60Asn Arg Asp Gly Asp Ser Tyr Arg Ser Pro Trp Ser Asn Lys Tyr Glu65 70 75 80Pro Pro Ile Asp Asp Gly Ala Met Pro Ser Ala Arg Leu Arg Lys Leu 85 90 95Glu Val Glu Ala Asn Asn Ala Phe Asp Gln Tyr Arg Asp Leu Tyr Phe 100 105 110Glu Gly Gly Val Ser Ser Val Tyr Leu Trp Asp Leu Asp His Gly Phe 115 120 125Ala Gly Val Ile Leu Ile Lys Lys Ala Gly Asp Gly Ser Lys Lys Ile 130 135 140Lys Gly Cys Trp Asp Ser Ile His Val Val Glu Val Gln Glu Lys Ser145 150 155 160Ser Gly Arg Thr Ala His Tyr Lys Leu Thr Ser Thr Val Met Leu Trp 165 170 175Leu Gln Thr Thr Lys Ala Gly Ser Gly Thr Met Asn Leu Gly Gly Ser 180 185 190Leu Thr Arg Gln Met Glu Lys Asp Glu Thr Val Gly Glu Ser Ser Pro 195 200 205His Ile Ala Asn Ile Gly Arg Leu Val Glu Asp Met Glu Asn Lys Ile 210 215 220Arg Ser Thr Leu Asn Glu Ile Tyr Phe Gly Lys Thr Lys Asp Ile Val225 230 235 240Asn Gly Leu Arg Ser Ile Asp Ser Leu Pro Asp Asn Gln Lys Tyr Arg 245 250 255Gln Leu Gln Lys Glu Leu Ser Gln Val Leu Thr Gln Arg Gln Ile Phe 260 265 270Ile Asp6224PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 62Asp Glu Thr Val Gly Glu Ser Ser Pro His Ile Ala Asn Ile Gly Arg1 5 10 15Leu Val Glu Asp Met Glu Asn Lys 206317PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 63Asp Ile Val Asn Gly Leu Arg Ser Ile Asp Ser Leu Pro Asp Asn Gln1 5 10 15Lys6414PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 64Glu Leu Ser Gln Val Leu Thr Gln Arg Gln Ile Phe Ile Asp1 5 106512PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 65Ile Arg Ser Thr Leu Asn Glu Ile Tyr Phe Gly Lys1 5 106613PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 66Leu Thr Ser Thr Val Met Leu Trp Leu Gln Thr Thr Lys1 5 106710PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 67Ser Ile Asp Ser Leu Pro Asp Asn Gln Lys1 5 106817PRTArtificial SequenceCapping protein (Actin filament) muscle Z-line beta fragment 68Tyr Glu Pro Pro Ile Asp Asp Gly Ala Met Pro Ser Ala Arg Leu Arg1 5 10 15Lys695799DNAArtificial Sequencemyosin heavy chain, fast skeletal muscle-like 69atgagtacgg acgcggagat gcaagtctac ggcaaggctg ccatatacct tcgtaagtct 60gagaaggaga ggatggaggc acaagccatg ccctttgatt caaagaacgc ctgctatgtg 120acagacaagg tggagctgta ccttaagggt ttagtcactg ccagggccga cgggaagtgt 180actgtgacag ctttagactt tcgtgaagga aaagagttca aagatgcaga catctatgag 240atgaaccccc ctaagtacga caagattgag gacatggcca tgatgaccta cctgaatgaa 300gcctctgtgt tgtataacct caaagagcgt tatgcagcat ggatgatcta tacctactct 360gggctcttct gtgccacggt gaacccctac aagtggctcc cagtgtacga cgaagaggtt 420gtcaacgcct acagagggaa gaagagggtg gaggctccac cacatatctt ctccgtctct 480gacaacgcct ttcagttcat gatgattgat aaggagaacc agtccgtcct gattactgga 540gaatccggtg caggaaagac tgtcaacacc aagcgtgtca tccagtactt tgccaccatt 600gcagtgtctg gtggcaagaa ggaagcagac cccaacaaaa tgcaggggtc tcttgaggat 660cagatcattg cagctaaccc tctgctagag tcttacggta atgccaagac agtgaggaac 720gacaactcgt ctcgctttgg taaattcatc aggattcact tccaagctgg taaactggct 780aaagctgaca ttgagaccta cctgctggag aagtccagag tgtccttcca actgcccgat 840gagagaggct accacatctt cttccagatg atgacaggcc acaaacctga gctagttgaa 900ttggcactcc tcaccaccaa cccctacgac ttccccatgt gcagccaggg tcagattgct 960gtggccagca tcaacgacaa tgaagagctg gatgccacag atgaagccat tacaatcctg 1020ggcttcacta atgaggagaa gcttggaata tacaagctga caggagctgt agtgcaccat 1080ggcaacttga aattcaagca gaagcagcgt gaggagcagg ccgagccaga cggcacagag 1140gtggctgata aaatcgccta cctgctgggc ctgaactcag ctgagatgtt gaaagctctg 1200tgctacccca gagtgaaggt cggcaacgag tatgtgacca agggacagac tgtggctcag 1260gttaataact cagtcagtgc tctggccaag tccatctatg agaggatgtt cttgtggatg 1320gtcatccgta tcaatgagat gttggacacc aagaatccaa ggcagttcta tatcggtgtg 1380cttgacattg ccgggtttga gatctttgat tacaacagca tggagcagct gtgcatcaac 1440ttcaccaatg agaaactgca acagtttttc aaccacacca tgttcgtcct ggagcaagag 1500gagtacaaga aggagggaat cgtctgggcc ttcattgact tcggcatgga tttggctgcc 1560tgcattgagc ttattgagaa gccattgggc atcttctcca tccttgaaga ggagtgcatg 1620ttccccaagt cttcagacac taccttcaag gacaagctgt acgcccagca tcttggtaaa 1680acaaaggcgt ttgagaagcc caagcctgcc aaaggcaagg cagaggccca cttctccctg 1740gtgcattacg ccggtactgt ggactacaac atcactggct ggctggagaa gaacaaggac 1800cccttgaacg actcagtttg tcaactgtat gggaagtccg gagtcaaaat tttggctgcc 1860ctgtatcccc ctccccctcc tgaggataaa gccaagaaag gaggcaagaa gaagggtggt 1920tccatgcaga ctgtgtcctc ccagttcagg gagaacttac ataagctgat gaccaacttg 1980aggagcactc atcctcactt tgtgcgctgc ctgatcccca acgagtcaaa gactccaggt 2040ctgatggaga acttcctggt tatccaccag ctcaggtgta atggtgtact ggagggtatc 2100aggatctgca gaaagggctt ccccagcaga atcatctatg ctgacttcaa gcaaaggtac 2160aaagtactga atgccagcgt catccctgag ggccagttca tggacaacaa gaaggcttct 2220gagaagctgc ttggatccat tgatgtgaat cacgaggatt acaagtttgg acacaccaag 2280gtgttcttca aagccggtct gctgggtgtc ctggaggaga tgagagatga gaagctggcc 2340tctctagtcg gcatgctcca ggctctcagc cgtggattcc tcatgaggag agagttcacc 2400aagatgatgg agaggagaga atcaatttac tccatccagt acaacatccg ctcattcatg 2460aatgtgaaaa cctggccatg gatgaagttg tacttcaaga tcaagcccct gctgcagagc 2520gctgagactg agaaggagct ggccaacatg aaggagaact atgagaaaat gaagacagac 2580ctggccaagg ctctgtctac aaagaagcaa atggaggaga agttggtgtc cctgacgcag 2640gagaagaacg acctggcact ccaagtcgca tctgaaggag agagtctgaa cgatgctgag 2700gaaaggtgcg aggggctcat caagagcaag atccagcagg aggccaaact caaagagacg 2760accgagaggc tggaggatga ggaggagatc aatgctgagt tgactgccaa gaagaggaag 2820ctggaggatg agtgctctga gctgaagaag gacattgatg atctggagct caccctggcc 2880aaagtggaga aggagaagca cgccactgaa aacaaggtta aaaacctgac agaggagatg 2940gcgtctatgg atgagagtgt tgccaagctg accaaggaga agaaagccct acaagaggcc 3000caccagcaga cactggatga cctgcaggca gaggaggaca aagtcaacac tctgaccaag 3060gccaagacca agctggaaca gcaagtggac gaccttgagg gttctctgga gcaagagaag 3120aagctccgca tggaccttga gagatccaag agaaagctgg agggagatct gaaactggcc 3180caggagtcca taatggacct ggagaatgac aagcagcaag ctgatgagaa aatcaagaag 3240aaggagtttg agaccactca gctcctcagc aaggttgagg atgagcagtc tctgggagct 3300cagctgcaga agaagatcaa ggaactccag gcccgtattg aggaactgga ggaggaaatt 3360gaggctgagc gtgctgccag ggctaaggtt gagaagcaga gggctgatct ctccagggaa 3420cttgaggaga tcagcgagag gctggaggag gccggaggcg ccactgctgc tcagattgag 3480atgaacaaga agcgtgaggc tgagttccag aagctgcgtc gtgatcttga agagtccacc 3540ctgcagcatg aggccacagc cgccgctctg cgcaagaagc aggccgacag tgtggctgag 3600ctcggggagc agatcgacaa cctgcagcgc gtcaagcaga agctggagaa ggagaagagc 3660gagtacaaga tggagattga tgacctctcc agcaacatgg aggccgttgc caaggctaag 3720ggcaatctag agaagatgtg ccgtactctt gaggaccagc tgagcgagct caagactaag 3780aatgatgaga atgttcgcca ggtcaacgac atcagcggac agagggccag actcctgaca 3840gaaaatggtg agtttggtag gcagctggag gagaaggaag ccctggtgtc tcagctgacc 3900agaggcaaac aggccttcac ccagcaggtg gaggagctga agagggcgat tgaggaggag 3960gtcaaggcta aaaatgcact ggcccacgga gttcagtctg cccgccatga ctgtgacctc 4020ctgagggagc agtttgagga ggagcaggag gccaaggcag agctgcaacg cggcatgtcc 4080aaggccaata gtgaggtggc tcagtggagg actaagtatg aaactgatgc catccagcgc 4140acagaggagc tggaggaggc caagaagaag ctggcccagc gtctgcagga tgccgaggag 4200accattgagg cgaccaactc caagtgcgcc tccctggaga agaccaagca gagactacag 4260ggagaggtgg aggacctcat gattgatgtt gagagagcca acgcattggc cgccaacctc 4320gacaagaagc agaggaactt tgacaaggtt ctggcagagt ggaagcagaa gtatgaggag 4380ggtcaggctg agctggaagg agctcagaag gaggctcgct ctatgagcac tgagctcttc 4440aagatgaaga actcctacga ggaggctctg gatcatctgg agactctgaa gagagagaac 4500aagaacctgc aacaggagat ctctgacctt actgagcaga ttggagagac tggcaagagc 4560atccatgagc tggagaaggc caagaagacc gtggagacag agaagtctga gatccagacc 4620gctctggagg aggctgaggg cacactggag cacgaggaat ccaagattct gcgtgtgcag 4680ctggagctga accagatcaa gggtgaggtg gacaggaaga tcgctgagaa ggacgaggag 4740atggagcaga tcaagaggaa cagccagagg atggttgact ccatgcagag caccctggac 4800tctgaggtca ggagcaggaa tgatgccctg agggtgaaga agaagatgga gggagacctg 4860aacgagatgg agatccagct gagccactcc aacaggcagg ccgctgaggc ccagaaacag 4920ctgaggaatg tccagggaca gctcaaggat gcccaattgc accttgatga tgccgtccgt 4980gtcgcagagg acatgaagga gcaggcagcc atggtggagc gcagaaacgg tctgatggtg 5040gctgagatcg aggagctgag agttgctctg gagcagacag agagaggccg caaagtggct 5100gagactgagc tggtagacgc cagcgagcgt gttggactgc tgcactccca gaacaccagc 5160cttctgaaca ccaagaagaa gctggagact gacctggtgc aggtgcaggg agaggtggat 5220gacatcgtcc aggaggccag gaatgcagaa gagaaggcca agaaggcaat cactgatgcg 5280gcaatgatgg ctgaggagct gaagaaggag caggacacca gctctcacct ggagagaatg 5340aagaagaacc tggagatcac agtcaaggac ctgcagcacc gcctggatga ggctgagaat 5400ctggccatga agggaggcaa gaaacaactc cagaaactgg aatccagggt gcgtgagctt 5460gagactgagg tggaggctga gcagagaaga ggtgtagacg cggtaaaggg agtccgcaag 5520tatgagcgca gagtcaagga gctcacttac cagactgagg aggataagaa gaatgttaac 5580agacttcagg acctggtaga taagctgcag atgaaagtga aggcctacaa gaggcacgct 5640gaggaagcgg aggaagcagc aaaccagcac atgtctaagt tcaggaaggt tcagcatgag 5700ctggaggagg ctgaggagcg tgctgacatc gctgagactc aggtcaacaa gctcagagcc 5760aagacccgtg actctggaaa gggaaaagaa gttgctgaa 5799701933PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like 70Met Ser Thr Asp Ala Glu Met Gln Val Tyr Gly Lys Ala Ala Ile Tyr1 5 10 15Leu Arg Lys Ser Glu Lys Glu Arg Met Glu Ala Gln Ala Met Pro Phe 20 25 30Asp Ser Lys Asn Ala Cys Tyr Val Thr Asp Lys Val Glu Leu Tyr Leu 35 40 45Lys Gly Leu Val Thr Ala Arg Ala Asp Gly Lys Cys Thr Val Thr Ala 50 55 60Leu Asp Phe Arg Glu Gly Lys Glu Phe Lys Asp Ala Asp Ile Tyr Glu65 70 75 80Met Asn Pro Pro Lys Tyr Asp Lys Ile Glu Asp Met Ala Met Met Thr 85 90 95Tyr Leu Asn Glu Ala Ser Val Leu Tyr Asn Leu Lys Glu Arg Tyr Ala 100 105 110Ala Trp Met Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Ala Thr Val Asn 115 120 125Pro Tyr Lys Trp Leu Pro Val Tyr Asp Glu Glu Val Val Asn Ala Tyr 130 135 140Arg Gly Lys Lys Arg Val Glu Ala Pro Pro His Ile Phe Ser Val Ser145 150 155 160Asp Asn Ala Phe Gln Phe Met Met Ile Asp Lys Glu Asn Gln Ser Val 165 170 175Leu Ile Thr Gly Glu Ser Gly Ala Gly Lys Thr Val Asn Thr Lys Arg 180 185 190Val Ile Gln Tyr Phe Ala Thr Ile Ala Val Ser Gly Gly Lys Lys Glu 195 200 205Ala Asp Pro Asn Lys Met Gln Gly Ser Leu Glu Asp Gln Ile Ile Ala 210 215 220Ala Asn Pro Leu Leu Glu Ser Tyr Gly Asn Ala Lys Thr Val Arg Asn225 230 235 240Asp Asn Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile His Phe Gln Ala 245 250 255Gly Lys Leu Ala Lys Ala Asp Ile Glu Thr Tyr Leu Leu Glu Lys Ser 260 265 270Arg Val Ser Phe Gln Leu Pro Asp Glu Arg Gly Tyr His Ile Phe Phe 275 280 285Gln Met Met Thr Gly His Lys Pro Glu Leu Val Glu Leu Ala Leu Leu 290 295 300Thr Thr Asn Pro Tyr Asp Phe Pro Met Cys Ser Gln Gly Gln Ile Ala305 310 315 320Val Ala Ser Ile Asn Asp Asn Glu Glu Leu Asp Ala Thr Asp Glu Ala 325 330 335Ile Thr Ile Leu Gly Phe Thr Asn Glu Glu Lys Leu Gly Ile Tyr Lys 340 345 350Leu Thr Gly Ala Val Val His His Gly Asn Leu Lys Phe Lys Gln Lys 355 360 365Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Val Ala Asp Lys 370 375 380Ile Ala Tyr Leu Leu Gly Leu Asn Ser Ala Glu Met Leu Lys Ala Leu385 390 395 400Cys Tyr Pro Arg Val Lys Val Gly Asn Glu Tyr Val Thr Lys Gly Gln 405 410 415Thr Val Ala Gln Val Asn Asn Ser Val Ser Ala Leu Ala Lys Ser Ile 420 425 430Tyr Glu Arg Met Phe Leu Trp Met Val Ile Arg Ile Asn Glu Met Leu 435 440 445Asp Thr Lys Asn Pro Arg Gln Phe Tyr Ile Gly Val Leu Asp Ile Ala 450 455 460Gly Phe Glu Ile Phe Asp Tyr Asn Ser Met Glu Gln

Leu Cys Ile Asn465 470 475 480Phe Thr Asn Glu Lys Leu Gln Gln Phe Phe Asn His Thr Met Phe Val 485 490 495Leu Glu Gln Glu Glu Tyr Lys Lys Glu Gly Ile Val Trp Ala Phe Ile 500 505 510Asp Phe Gly Met Asp Leu Ala Ala Cys Ile Glu Leu Ile Glu Lys Pro 515 520 525Leu Gly Ile Phe Ser Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ser 530 535 540Ser Asp Thr Thr Phe Lys Asp Lys Leu Tyr Ala Gln His Leu Gly Lys545 550 555 560Thr Lys Ala Phe Glu Lys Pro Lys Pro Ala Lys Gly Lys Ala Glu Ala 565 570 575His Phe Ser Leu Val His Tyr Ala Gly Thr Val Asp Tyr Asn Ile Thr 580 585 590Gly Trp Leu Glu Lys Asn Lys Asp Pro Leu Asn Asp Ser Val Cys Gln 595 600 605Leu Tyr Gly Lys Ser Gly Val Lys Ile Leu Ala Ala Leu Tyr Pro Pro 610 615 620Pro Pro Pro Glu Asp Lys Ala Lys Lys Gly Gly Lys Lys Lys Gly Gly625 630 635 640Ser Met Gln Thr Val Ser Ser Gln Phe Arg Glu Asn Leu His Lys Leu 645 650 655Met Thr Asn Leu Arg Ser Thr His Pro His Phe Val Arg Cys Leu Ile 660 665 670Pro Asn Glu Ser Lys Thr Pro Gly Leu Met Glu Asn Phe Leu Val Ile 675 680 685His Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg Ile Cys Arg 690 695 700Lys Gly Phe Pro Ser Arg Ile Ile Tyr Ala Asp Phe Lys Gln Arg Tyr705 710 715 720Lys Val Leu Asn Ala Ser Val Ile Pro Glu Gly Gln Phe Met Asp Asn 725 730 735Lys Lys Ala Ser Glu Lys Leu Leu Gly Ser Ile Asp Val Asn His Glu 740 745 750Asp Tyr Lys Phe Gly His Thr Lys Val Phe Phe Lys Ala Gly Leu Leu 755 760 765Gly Val Leu Glu Glu Met Arg Asp Glu Lys Leu Ala Ser Leu Val Gly 770 775 780Met Leu Gln Ala Leu Ser Arg Gly Phe Leu Met Arg Arg Glu Phe Thr785 790 795 800Lys Met Met Glu Arg Arg Glu Ser Ile Tyr Ser Ile Gln Tyr Asn Ile 805 810 815Arg Ser Phe Met Asn Val Lys Thr Trp Pro Trp Met Lys Leu Tyr Phe 820 825 830Lys Ile Lys Pro Leu Leu Gln Ser Ala Glu Thr Glu Lys Glu Leu Ala 835 840 845Asn Met Lys Glu Asn Tyr Glu Lys Met Lys Thr Asp Leu Ala Lys Ala 850 855 860Leu Ser Thr Lys Lys Gln Met Glu Glu Lys Leu Val Ser Leu Thr Gln865 870 875 880Glu Lys Asn Asp Leu Ala Leu Gln Val Ala Ser Glu Gly Glu Ser Leu 885 890 895Asn Asp Ala Glu Glu Arg Cys Glu Gly Leu Ile Lys Ser Lys Ile Gln 900 905 910Gln Glu Ala Lys Leu Lys Glu Thr Thr Glu Arg Leu Glu Asp Glu Glu 915 920 925Glu Ile Asn Ala Glu Leu Thr Ala Lys Lys Arg Lys Leu Glu Asp Glu 930 935 940Cys Ser Glu Leu Lys Lys Asp Ile Asp Asp Leu Glu Leu Thr Leu Ala945 950 955 960Lys Val Glu Lys Glu Lys His Ala Thr Glu Asn Lys Val Lys Asn Leu 965 970 975Thr Glu Glu Met Ala Ser Met Asp Glu Ser Val Ala Lys Leu Thr Lys 980 985 990Glu Lys Lys Ala Leu Gln Glu Ala His Gln Gln Thr Leu Asp Asp Leu 995 1000 1005Gln Ala Glu Glu Asp Lys Val Asn Thr Leu Thr Lys Ala Lys Thr 1010 1015 1020Lys Leu Glu Gln Gln Val Asp Asp Leu Glu Gly Ser Leu Glu Gln 1025 1030 1035Glu Lys Lys Leu Arg Met Asp Leu Glu Arg Ser Lys Arg Lys Leu 1040 1045 1050Glu Gly Asp Leu Lys Leu Ala Gln Glu Ser Ile Met Asp Leu Glu 1055 1060 1065Asn Asp Lys Gln Gln Ala Asp Glu Lys Ile Lys Lys Lys Glu Phe 1070 1075 1080Glu Thr Thr Gln Leu Leu Ser Lys Val Glu Asp Glu Gln Ser Leu 1085 1090 1095Gly Ala Gln Leu Gln Lys Lys Ile Lys Glu Leu Gln Ala Arg Ile 1100 1105 1110Glu Glu Leu Glu Glu Glu Ile Glu Ala Glu Arg Ala Ala Arg Ala 1115 1120 1125Lys Val Glu Lys Gln Arg Ala Asp Leu Ser Arg Glu Leu Glu Glu 1130 1135 1140Ile Ser Glu Arg Leu Glu Glu Ala Gly Gly Ala Thr Ala Ala Gln 1145 1150 1155Ile Glu Met Asn Lys Lys Arg Glu Ala Glu Phe Gln Lys Leu Arg 1160 1165 1170Arg Asp Leu Glu Glu Ser Thr Leu Gln His Glu Ala Thr Ala Ala 1175 1180 1185Ala Leu Arg Lys Lys Gln Ala Asp Ser Val Ala Glu Leu Gly Glu 1190 1195 1200Gln Ile Asp Asn Leu Gln Arg Val Lys Gln Lys Leu Glu Lys Glu 1205 1210 1215Lys Ser Glu Tyr Lys Met Glu Ile Asp Asp Leu Ser Ser Asn Met 1220 1225 1230Glu Ala Val Ala Lys Ala Lys Gly Asn Leu Glu Lys Met Cys Arg 1235 1240 1245Thr Leu Glu Asp Gln Leu Ser Glu Leu Lys Thr Lys Asn Asp Glu 1250 1255 1260Asn Val Arg Gln Val Asn Asp Ile Ser Gly Gln Arg Ala Arg Leu 1265 1270 1275Leu Thr Glu Asn Gly Glu Phe Gly Arg Gln Leu Glu Glu Lys Glu 1280 1285 1290Ala Leu Val Ser Gln Leu Thr Arg Gly Lys Gln Ala Phe Thr Gln 1295 1300 1305Gln Val Glu Glu Leu Lys Arg Ala Ile Glu Glu Glu Val Lys Ala 1310 1315 1320Lys Asn Ala Leu Ala His Gly Val Gln Ser Ala Arg His Asp Cys 1325 1330 1335Asp Leu Leu Arg Glu Gln Phe Glu Glu Glu Gln Glu Ala Lys Ala 1340 1345 1350Glu Leu Gln Arg Gly Met Ser Lys Ala Asn Ser Glu Val Ala Gln 1355 1360 1365Trp Arg Thr Lys Tyr Glu Thr Asp Ala Ile Gln Arg Thr Glu Glu 1370 1375 1380Leu Glu Glu Ala Lys Lys Lys Leu Ala Gln Arg Leu Gln Asp Ala 1385 1390 1395Glu Glu Thr Ile Glu Ala Thr Asn Ser Lys Cys Ala Ser Leu Glu 1400 1405 1410Lys Thr Lys Gln Arg Leu Gln Gly Glu Val Glu Asp Leu Met Ile 1415 1420 1425Asp Val Glu Arg Ala Asn Ala Leu Ala Ala Asn Leu Asp Lys Lys 1430 1435 1440Gln Arg Asn Phe Asp Lys Val Leu Ala Glu Trp Lys Gln Lys Tyr 1445 1450 1455Glu Glu Gly Gln Ala Glu Leu Glu Gly Ala Gln Lys Glu Ala Arg 1460 1465 1470Ser Met Ser Thr Glu Leu Phe Lys Met Lys Asn Ser Tyr Glu Glu 1475 1480 1485Ala Leu Asp His Leu Glu Thr Leu Lys Arg Glu Asn Lys Asn Leu 1490 1495 1500Gln Gln Glu Ile Ser Asp Leu Thr Glu Gln Ile Gly Glu Thr Gly 1505 1510 1515Lys Ser Ile His Glu Leu Glu Lys Ala Lys Lys Thr Val Glu Thr 1520 1525 1530Glu Lys Ser Glu Ile Gln Thr Ala Leu Glu Glu Ala Glu Gly Thr 1535 1540 1545Leu Glu His Glu Glu Ser Lys Ile Leu Arg Val Gln Leu Glu Leu 1550 1555 1560Asn Gln Ile Lys Gly Glu Val Asp Arg Lys Ile Ala Glu Lys Asp 1565 1570 1575Glu Glu Met Glu Gln Ile Lys Arg Asn Ser Gln Arg Met Val Asp 1580 1585 1590Ser Met Gln Ser Thr Leu Asp Ser Glu Val Arg Ser Arg Asn Asp 1595 1600 1605Ala Leu Arg Val Lys Lys Lys Met Glu Gly Asp Leu Asn Glu Met 1610 1615 1620Glu Ile Gln Leu Ser His Ser Asn Arg Gln Ala Ala Glu Ala Gln 1625 1630 1635Lys Gln Leu Arg Asn Val Gln Gly Gln Leu Lys Asp Ala Gln Leu 1640 1645 1650His Leu Asp Asp Ala Val Arg Val Ala Glu Asp Met Lys Glu Gln 1655 1660 1665Ala Ala Met Val Glu Arg Arg Asn Gly Leu Met Val Ala Glu Ile 1670 1675 1680Glu Glu Leu Arg Val Ala Leu Glu Gln Thr Glu Arg Gly Arg Lys 1685 1690 1695Val Ala Glu Thr Glu Leu Val Asp Ala Ser Glu Arg Val Gly Leu 1700 1705 1710Leu His Ser Gln Asn Thr Ser Leu Leu Asn Thr Lys Lys Lys Leu 1715 1720 1725Glu Thr Asp Leu Val Gln Val Gln Gly Glu Val Asp Asp Ile Val 1730 1735 1740Gln Glu Ala Arg Asn Ala Glu Glu Lys Ala Lys Lys Ala Ile Thr 1745 1750 1755Asp Ala Ala Met Met Ala Glu Glu Leu Lys Lys Glu Gln Asp Thr 1760 1765 1770Ser Ser His Leu Glu Arg Met Lys Lys Asn Leu Glu Ile Thr Val 1775 1780 1785Lys Asp Leu Gln His Arg Leu Asp Glu Ala Glu Asn Leu Ala Met 1790 1795 1800Lys Gly Gly Lys Lys Gln Leu Gln Lys Leu Glu Ser Arg Val Arg 1805 1810 1815Glu Leu Glu Thr Glu Val Glu Ala Glu Gln Arg Arg Gly Val Asp 1820 1825 1830Ala Val Lys Gly Val Arg Lys Tyr Glu Arg Arg Val Lys Glu Leu 1835 1840 1845Thr Tyr Gln Thr Glu Glu Asp Lys Lys Asn Val Asn Arg Leu Gln 1850 1855 1860Asp Leu Val Asp Lys Leu Gln Met Lys Val Lys Ala Tyr Lys Arg 1865 1870 1875His Ala Glu Glu Ala Glu Glu Ala Ala Asn Gln His Met Ser Lys 1880 1885 1890Phe Arg Lys Val Gln His Glu Leu Glu Glu Ala Glu Glu Arg Ala 1895 1900 1905Asp Ile Ala Glu Thr Gln Val Asn Lys Leu Arg Ala Lys Thr Arg 1910 1915 1920Asp Ser Gly Lys Gly Lys Glu Val Ala Glu 1925 1930717PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 71Ala Ala Ile Tyr Leu Arg Lys1 57210PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 72Ala Asp Ile Glu Thr Tyr Leu Leu Glu Lys1 5 107319PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 73Ala Leu Gln Glu Ala His Gln Gln Thr Leu Asp Asp Leu Gln Ala Glu1 5 10 15Glu Asp Lys7411PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 74Asp Ile Asp Asp Leu Glu Leu Thr Leu Ala Lys1 5 107511PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 75Glu Ala Leu Val Ser Gln Leu Thr Arg Gly Lys1 5 107610PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 76Glu Phe Glu Thr Thr Gln Leu Leu Ser Lys1 5 107710PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 77Glu Leu Thr Tyr Gln Thr Glu Glu Asp Lys1 5 107815PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 78Glu Asn Gln Ser Val Leu Ile Thr Gly Glu Ser Gly Ala Gly Lys1 5 10 157919PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 79Glu Thr Thr Glu Arg Leu Glu Asp Glu Glu Glu Ile Asn Ala Glu Leu1 5 10 15Thr Ala Lys8010PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 80Phe Ile Arg Ile His Phe Gln Ala Gly Lys1 5 108112PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 81Gly Phe Pro Ser Arg Ile Ile Tyr Ala Asp Phe Lys1 5 108210PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 82Gly Leu Val Thr Ala Arg Ala Asp Gly Lys1 5 108316PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 83Gly Gln Thr Val Ala Gln Val Asn Asn Ser Val Ser Ala Leu Ala Lys1 5 10 158412PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 84Ile Lys Pro Leu Leu Gln Ser Ala Glu Thr Glu Lys1 5 108514PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 85Ile Leu Ala Ala Leu Tyr Pro Pro Pro Pro Pro Glu Asp Lys1 5 108612PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 86Ile Leu Arg Val Gln Leu Glu Leu Asn Gln Ile Lys1 5 108718PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 87Leu Ala Gln Arg Leu Gln Asp Ala Glu Glu Thr Ile Glu Ala Thr Asn1 5 10 15Ser Lys8816PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 88Leu Glu Gln Gln Val Asp Asp Leu Glu Gly Ser Leu Glu Gln Glu Lys1 5 10 158924PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 89Leu Glu Ser Arg Val Arg Glu Leu Glu Thr Glu Val Glu Ala Glu Gln1 5 10 15Arg Arg Gly Val Asp Ala Val Lys 209013PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 90Leu Leu Gly Ser Ile Asp Val Asn His Glu Asp Tyr Lys1 5 109121PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 91Leu Arg Arg Asp Leu Glu Glu Ser Thr Leu Gln His Glu Ala Thr Ala1 5 10 15Ala Ala Leu Arg Lys 209212PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 92Leu Thr Gly Ala Val Val His His Gly Asn Leu Lys1 5 109332PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 93Asn Asp Glu Asn Val Arg Gln Val Asn Asp Ile Ser Gly Gln Arg Ala1 5 10 15Arg Leu Leu Thr Glu Asn Gly Glu Phe Gly Arg Gln Leu Glu Glu Lys 20 25 30947PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 94Asn Leu Glu Ile Thr Val Lys1 59518PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 95Asn Leu Gln Gln Glu Ile Ser Asp Leu Thr Glu Gln Ile Gly Glu Thr1 5 10 15Gly Lys9614PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 96Asn Ser Tyr Glu Glu Ala Leu Asp His Leu Glu Thr Leu Lys1 5 109719PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 97Gln Ala Asp Ser Val Ala Glu Leu Gly Glu Gln Ile Asp Asn Leu Gln1 5 10 15Arg Val Lys9811PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 98Gln Ala Phe Thr Gln Gln Val Glu Glu Leu Lys1 5 109916PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 99Gln Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Val Ala Asp Lys1 5 10 151008PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 100Arg Ala Ile Glu Glu Glu Val Lys1 510120PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 101Ser Glu Ile Gln Thr Ala Leu Glu Glu Ala Glu Gly Thr Leu Glu His1 5 10 15Glu Glu Ser Lys 2010212PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 102Thr Val Arg Asn Asp Asn Ser Ser Arg Phe Gly Lys1 5 1010327PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 103Val Ala Glu Thr Glu Leu Val Asp Ala Ser Glu Arg Val Gly Leu Leu1 5 10 15His Ser Gln Asn Thr Ser Leu Leu Asn Thr Lys 20 2510413PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 104Val Glu Asp Glu Gln Ser Leu Gly Ala Gln Leu Gln Lys1 5 101058PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 105Val Gly Asn Glu Tyr Val Thr Lys1 510621PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 106Val Gln His Glu Leu Glu Glu Ala Glu Glu Arg Ala Asp Ile Ala Glu1 5 10 15Thr Gln Val Asn Lys 2010713PRTArtificial Sequencemyosin heavy chain, fast skeletal muscle-like fragment 107Tyr Glu Glu Gly Gln Ala Glu Leu Glu Gly Ala Gln Lys1 5 1010816PRTArtificial

Sequencemyosin heavy chain, fast skeletal muscle-like fragment 108Tyr Glu Thr Asp Ala Ile Gln Arg Thr Glu Glu Leu Glu Glu Ala Lys1 5 10 151092532DNAArtificial Sequenceglycogen phosphorylase, muscle form-like 109atgtcaaaac cattgtcaga ccacgataaa aggaagcaga tctccgtgag aggtcttgct 60ggtgtggaaa atgtcgcaga actgaaggtc gctttcaaca ggcatctcca ttttacactg 120gtcaaggaca gaaatgtggc aaacaaacgg gattactact ttgctctcgc caacaccgtg 180cgtgaccact tggtgggcag gtggatcaga acccagcagt actactatga gaaagatccc 240aaacgtgtgt actacatctc cctggagttt tatatgggtc gcaccctgca gaacactatg 300gtgaacctgg ccctggagaa cgcttgtgat gaggccatat accagctggg tctggacatg 360gaggagctgg aggacatgga agaggatgct ggcctgggaa acggtggtct tggccgtctt 420gccgcatgct tcctggactc aatggcttct ctaggccttg ctgcctatgg ctatggtatc 480cgctatgagt ttggcatctt caatcagaag atcgtcaatg gctggcaggt tgaggaggcc 540gatgattggt tgcgttacgg caacccctgg gagaaggccc gccctgagta catgcgcccc 600gtcaagttct atggcaggac cgagcacacc ccagatggtg tgaaatgggt tgacactcag 660gtagtgttgg ctctgccata tgacacccct attcccggct acagaaacaa cattgtcaac 720accatgagac tgtggtctgc taaggcccca tgcgacttca acctgaaaga cttcaacgtt 780ggtggctaca ttcaggctgt gttggacaga aacttgtgtg agaacatttc ccgcgtgctg 840taccccaatg ataatttctt tgagggcaag gagctgcgtc tgaagcagga gtactttgtg 900gtggccgcca ctctgcagga cgtcgtccgt cgtttcaagg cctctaagtt tggctccaga 960gagattgtcc gcacagactt cgccgagcta ccaaacaaag ttgccatcca gctgaatgac 1020actcaccctg ccatggctat tcctgagctg atgagggtcc tggttgatga ggagaagctg 1080ggttgggaca aggcctggga cgtgtgtgtc cgtacctgtg cctacacaaa ccacaccgtg 1140ctgcctgagg ccctggagcg ctggcccatt gacctgttcc atcacctgct gccacgtcac 1200ctggagatca tctacgagat caaccgtcgc ttcatggagt atgttgcctc gaagttccct 1260ggagacaacg accgtctgcg tcgcatgtcc ctgattgagg agggaggatg caagaaagtc 1320aacatggctc atctgtgtat cgttggttcc catgctgtca acggcgtggc ccgcatccac 1380tctgagatcc tcatcgccac tctgttcaag gacttctatg agttggaccc acacaagttt 1440cagaacaaga ccaatgggat caccccccgt cgctggctgg ttatgtgcaa ccccggcttg 1500gcggaggtca tcgcagagaa aattggagag gagtttatcc gtgaccttga ccagcttaag 1560cgactgttga agttcgttaa tgatgatgct ttcatccgtg acatcgccaa agtcaagcag 1620gagaacaagc tgaagttcgc tgtgcacctg gaagagcact acaaggtcaa gatcaacccc 1680cagtccatgt ttgacttcca agtcaaaaga atccacgagt acaaaagaca gctgctcaac 1740tgtctgcaca tgatcaccta ctacaaccgt atcaagaagg agcccaacaa gcactggacc 1800ccaagaacca tcatggtcgg aggaaaggct gcaccaggct accacacagc caagatgatc 1860atccgtctca tcacagctat cggtgaggtt gtcaaccacg accccgttgt cggcgaccgc 1920ctcaaagtta tcttcctgga gaactacaga gtcaccctgg ctgagaaagc catcccctct 1980gccgacctgt ctgagcagat ctctacagct ggcaccgagg cctctggcac tggtaacatg 2040aagttcatgc tgaacggtgc tcttaccatt ggcaccatgg atggagccaa tgtggagatg 2100gccgaggagg ccggagagga aaacttcttc atcttcggta tgagagtgga ggatgtggac 2160gcaatggacg ccggcaaagg ataccacgcc tctgagtact acaaccgtat tcccgagctg 2220aagcaggcca tggaccagat tgctggcggc ttcttcagtc ccaagcagcc tgacctcttc 2280aaggaacttg tggacctgct gatgcaccat gacaggttca aggtgtttgc tgactacgaa 2340gcctacatca aatgccagga caaggtcaac caactgtaca agaatcccaa ggaatggacc 2400aagatggtga tccataacat tgcgggctgt ggtaaattct ccagcgaccg caccattgcc 2460cagtacgccc gagagatctg gggcatggag cccagcctgg agaagatccc tgcccccgat 2520gagaaactca aa 2532110844PRTArtificial Sequenceglycogen phosphorylase, muscle form-like 110Met Ser Lys Pro Leu Ser Asp His Asp Lys Arg Lys Gln Ile Ser Val1 5 10 15Arg Gly Leu Ala Gly Val Glu Asn Val Ala Glu Leu Lys Val Ala Phe 20 25 30Asn Arg His Leu His Phe Thr Leu Val Lys Asp Arg Asn Val Ala Asn 35 40 45Lys Arg Asp Tyr Tyr Phe Ala Leu Ala Asn Thr Val Arg Asp His Leu 50 55 60Val Gly Arg Trp Ile Arg Thr Gln Gln Tyr Tyr Tyr Glu Lys Asp Pro65 70 75 80Lys Arg Val Tyr Tyr Ile Ser Leu Glu Phe Tyr Met Gly Arg Thr Leu 85 90 95Gln Asn Thr Met Val Asn Leu Ala Leu Glu Asn Ala Cys Asp Glu Ala 100 105 110Ile Tyr Gln Leu Gly Leu Asp Met Glu Glu Leu Glu Asp Met Glu Glu 115 120 125Asp Ala Gly Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ala Cys Phe 130 135 140Leu Asp Ser Met Ala Ser Leu Gly Leu Ala Ala Tyr Gly Tyr Gly Ile145 150 155 160Arg Tyr Glu Phe Gly Ile Phe Asn Gln Lys Ile Val Asn Gly Trp Gln 165 170 175Val Glu Glu Ala Asp Asp Trp Leu Arg Tyr Gly Asn Pro Trp Glu Lys 180 185 190Ala Arg Pro Glu Tyr Met Arg Pro Val Lys Phe Tyr Gly Arg Thr Glu 195 200 205His Thr Pro Asp Gly Val Lys Trp Val Asp Thr Gln Val Val Leu Ala 210 215 220Leu Pro Tyr Asp Thr Pro Ile Pro Gly Tyr Arg Asn Asn Ile Val Asn225 230 235 240Thr Met Arg Leu Trp Ser Ala Lys Ala Pro Cys Asp Phe Asn Leu Lys 245 250 255Asp Phe Asn Val Gly Gly Tyr Ile Gln Ala Val Leu Asp Arg Asn Leu 260 265 270Cys Glu Asn Ile Ser Arg Val Leu Tyr Pro Asn Asp Asn Phe Phe Glu 275 280 285Gly Lys Glu Leu Arg Leu Lys Gln Glu Tyr Phe Val Val Ala Ala Thr 290 295 300Leu Gln Asp Val Val Arg Arg Phe Lys Ala Ser Lys Phe Gly Ser Arg305 310 315 320Glu Ile Val Arg Thr Asp Phe Ala Glu Leu Pro Asn Lys Val Ala Ile 325 330 335Gln Leu Asn Asp Thr His Pro Ala Met Ala Ile Pro Glu Leu Met Arg 340 345 350Val Leu Val Asp Glu Glu Lys Leu Gly Trp Asp Lys Ala Trp Asp Val 355 360 365Cys Val Arg Thr Cys Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala 370 375 380Leu Glu Arg Trp Pro Ile Asp Leu Phe His His Leu Leu Pro Arg His385 390 395 400Leu Glu Ile Ile Tyr Glu Ile Asn Arg Arg Phe Met Glu Tyr Val Ala 405 410 415Ser Lys Phe Pro Gly Asp Asn Asp Arg Leu Arg Arg Met Ser Leu Ile 420 425 430Glu Glu Gly Gly Cys Lys Lys Val Asn Met Ala His Leu Cys Ile Val 435 440 445Gly Ser His Ala Val Asn Gly Val Ala Arg Ile His Ser Glu Ile Leu 450 455 460Ile Ala Thr Leu Phe Lys Asp Phe Tyr Glu Leu Asp Pro His Lys Phe465 470 475 480Gln Asn Lys Thr Asn Gly Ile Thr Pro Arg Arg Trp Leu Val Met Cys 485 490 495Asn Pro Gly Leu Ala Glu Val Ile Ala Glu Lys Ile Gly Glu Glu Phe 500 505 510Ile Arg Asp Leu Asp Gln Leu Lys Arg Leu Leu Lys Phe Val Asn Asp 515 520 525Asp Ala Phe Ile Arg Asp Ile Ala Lys Val Lys Gln Glu Asn Lys Leu 530 535 540Lys Phe Ala Val His Leu Glu Glu His Tyr Lys Val Lys Ile Asn Pro545 550 555 560Gln Ser Met Phe Asp Phe Gln Val Lys Arg Ile His Glu Tyr Lys Arg 565 570 575Gln Leu Leu Asn Cys Leu His Met Ile Thr Tyr Tyr Asn Arg Ile Lys 580 585 590Lys Glu Pro Asn Lys His Trp Thr Pro Arg Thr Ile Met Val Gly Gly 595 600 605Lys Ala Ala Pro Gly Tyr His Thr Ala Lys Met Ile Ile Arg Leu Ile 610 615 620Thr Ala Ile Gly Glu Val Val Asn His Asp Pro Val Val Gly Asp Arg625 630 635 640Leu Lys Val Ile Phe Leu Glu Asn Tyr Arg Val Thr Leu Ala Glu Lys 645 650 655Ala Ile Pro Ser Ala Asp Leu Ser Glu Gln Ile Ser Thr Ala Gly Thr 660 665 670Glu Ala Ser Gly Thr Gly Asn Met Lys Phe Met Leu Asn Gly Ala Leu 675 680 685Thr Ile Gly Thr Met Asp Gly Ala Asn Val Glu Met Ala Glu Glu Ala 690 695 700Gly Glu Glu Asn Phe Phe Ile Phe Gly Met Arg Val Glu Asp Val Asp705 710 715 720Ala Met Asp Ala Gly Lys Gly Tyr His Ala Ser Glu Tyr Tyr Asn Arg 725 730 735Ile Pro Glu Leu Lys Gln Ala Met Asp Gln Ile Ala Gly Gly Phe Phe 740 745 750Ser Pro Lys Gln Pro Asp Leu Phe Lys Glu Leu Val Asp Leu Leu Met 755 760 765His His Asp Arg Phe Lys Val Phe Ala Asp Tyr Glu Ala Tyr Ile Lys 770 775 780Cys Gln Asp Lys Val Asn Gln Leu Tyr Lys Asn Pro Lys Glu Trp Thr785 790 795 800Lys Met Val Ile His Asn Ile Ala Gly Cys Gly Lys Phe Ser Ser Asp 805 810 815Arg Thr Ile Ala Gln Tyr Ala Arg Glu Ile Trp Gly Met Glu Pro Ser 820 825 830Leu Glu Lys Ile Pro Ala Pro Asp Glu Lys Leu Lys 835 8401119PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 111Asp Phe Tyr Glu Leu Asp Pro His Lys1 511210PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 112Phe Ala Val His Leu Glu Glu His Tyr Lys1 5 1011313PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 113Phe Tyr Gly Arg Thr Glu His Thr Pro Asp Gly Val Lys1 5 1011415PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 114Gly Tyr His Ala Ser Glu Tyr Tyr Asn Arg Ile Pro Glu Leu Lys1 5 10 1511513PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 115Ile Gly Glu Glu Phe Ile Arg Asp Leu Asp Gln Leu Lys1 5 1011614PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 116Gln Ala Met Asp Gln Ile Ala Gly Gly Phe Phe Ser Pro Lys1 5 1011717PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 117Gln Ile Ser Val Arg Gly Leu Ala Gly Val Glu Asn Val Ala Glu Leu1 5 10 15Lys11810PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 118Val Phe Ala Asp Tyr Glu Ala Tyr Ile Lys1 5 1011914PRTArtificial Sequenceglycogen phosphorylase, muscle form-like fragment 119Val Ile Phe Leu Glu Asn Tyr Arg Val Thr Leu Ala Glu Lys1 5 101203399DNAArtificial Sequencemyosin-binding protein C, fast-type-like 120atgcctgaag ccgctgatgc agtgaagccg gagggcgagg agggagaagc accagcagaa 60gccgaagaaa atactgcaga agatgatgag ccactgcctg aaggagagga gggtgcccat 120cagtcacagg agattacagg actgtttctt cagcaccctg agactacggt tgccataaca 180ggtacggaca ttgtgtttac tgccaaagtg gactctacta cactatcaag gaaacccacc 240attaaatggc tgaaggggaa gtggctggac ctgggcagca aagcaggaaa acacatgcag 300ttcaaagaga cttttgacag agccacaaag atctacacct gggacatgaa gataatcaag 360gtggtccctg gtgacgctgg ggcctacagg tgtgaggtca cctccaaaga caagtgtgac 420agcagcgctt tcgacatctc cgtggaggct gtgcaaattg aggaggaaca tgattctttg 480gccgcattca aaagaacgga tgctggagag gatgaaggca gtttggattt cagtgctttg 540ctgaaagcta ccaagaagaa gaagaagcca gtcgtagatg aggagaaggc agacgtgtgg 600gaaatcctga agaacgctca acccagcgag tatgagaaga ttgcttttga gtacggcatt 660acagacctga ggggtctgct caagcgactg aagaagatga agactgttga gcccaagcac 720agcgacgctt tcctaaagag aatggagtct gcctactctg tggataaggg caagaaaatc 780gtactgcagg tggaagtggt tgacccaaat gcccaggtca aatggttgaa gaacggtcag 840gagataaaat catcagccaa gtacatcata gaatcagttg gcaacatcag gacgctcacc 900atcaacaagt gtagcctggc tgacgacgct gcgtacgagt gtgtgattgg ggaagagaag 960tccttcacag aggtgtttgt caaagagccc ccggtcacca tcaccaagct gctggatgat 1020taccacgtgg tggtgggaga gagagtggag tttgaggtgg aggtgtctgt ggagggagca 1080cacgttaact ggatgtttga ggaccaagaa ctctccaggg acacccataa gtaccgcttt 1140aagaaggacg ggttgaagca catgctcatc atccaagagg ctacgctgga tgacattgga 1200atgtactggt gcttcaccaa cggcgggcga accaaaggag agctagaagt tgaagagaaa 1260gaactggagg tgttgcagaa catcgctgac ctgacggtga aggccgagga ccaggccatg 1320ttcaagtgtg aggtgtctga tgagaaggta acagggaaat ggctcaaaga tggcgtggag 1380gtgctgccca gcagtcgcat caagctgacc cacatcggaa gaatccatcg gcttaccata 1440gacgacgtca agcccgagga cgctggagac tataccttta tcccagacgg atatgccctc 1500tcactgtccg ccaaactcaa cttcctggaa attaagatcg actatgtccc ccgccaagag 1560ccacccaaga tccatctaga caccaccggc aacatggttt cccagaacac catcattgtg 1620gtggctggca acaagctgcg cctggacgtg gagatctctg gagagcctcc tcccactgtc 1680gtctgggcaa agggagacac ggcaatcaca gctgtggagg ggcgtgtgag gactgagagc 1740cggaaggacc tgagctgctt cgtcatagag ggggcagaga gagaggatga gggcaactac 1800accatcattg tcaccaaccc tgccggagag gacaaggctc atctgtttgt caagattgtt 1860gatgtgcctg actgccctga gaatgtaaag tgtacctcgg tgggagaaga ctgtgccacc 1920atggtctggg acccacccaa gttcgacgga ggcgcaccag tcacaggcta tctcatggag 1980aggaagaaga agggctccac caggtggacg aagctcaact ttgacgtgta cgagggggtg 2040acgtacgaag ccaagaggat gattgagggc gtgctctacg agatgagagt gtacgccgtc 2100aatggcattg gcatgtccca gcccagcctc aattccaaac ccttcatgcc catcgctgca 2160accagtgagc ctctgggcct caaggtgcat gatgtcacag acaccacatg taccctgaag 2220tggctggccc ctgagaagat cggtgccgga ggcctggacg gatacgttat tgagtactgc 2280aaggagggag acacggagtg ggtggtggcg aacactgagc tggtggagag acagaattac 2340acagtccgca acctccccac cgcggagaag atcaacttca gagtggtggc cgtgaacatt 2400gctggacgca gcccccctac cggcctgagc cagcccgtca ctatccgcga gatcgtggaa 2460ctgcccaaga tccgcctccc tcgctacttg agacagaagt acattagaag agtgggcgac 2520aaaatcaacc tgaccatccc cttccagggt aagccacgtc ccaaagtgca atggtacaag 2580gatggggaag agcttgacac tcgagttgcc agcatacgca attccgaagt ggactctatc 2640ctgttcatcc gctcggctga gagatcccac tctgggaagt acgagttggt cctgcagatt 2700gagaacatgg aggccagggc tattctggaa atcaggatcg tagaggcacc tgggcctccc 2760gaggtagtga aggtcacaga cgtttggggc ttcaacgctg cgctggagtg gaagccaccg 2820aaggacgacg gcaactgcga gatcaccggt tacacaatac agaaggcgga caagaagaca 2880aatgaatggt tcactatcta cgagcacaac aggaggacaa actgcacagc ctcagacctg 2940atcatgggaa atgagtacat gttccgtgtc ttcagcgaga acctcgttgg aaagagtgag 3000gatttctgcc tcagcaagga cacagccatc atacctaaga taggattgga gtacaaccca 3060cctccattca aggagaagga tatgcaaagc gcccccaagt tcatacagcc cctgcttgac 3120aggtctgtgg tggcaggcta cagtaccacc atcagctctg ctgtcaagac ttgtcctaaa 3180cctaagatca ggtggttgag gaataagatt cccctggacg agaaccctag gttcctgatg 3240cagaacaacc agggggtttt gaccctgaac atccgcaagc ccagtcagta tgacgggggc 3300aagttcacct gcaaggctat caactctttg ggggaggacg tcgtggagtg caccctactc 3360gtacgagctc tcaaggacaa ggaagatgga gacgagaaa 33991211133PRTArtificial Sequencemyosin-binding protein C, fast-type-like 121Met Pro Glu Ala Ala Asp Ala Val Lys Pro Glu Gly Glu Glu Gly Glu1 5 10 15Ala Pro Ala Glu Ala Glu Glu Asn Thr Ala Glu Asp Asp Glu Pro Leu 20 25 30Pro Glu Gly Glu Glu Gly Ala His Gln Ser Gln Glu Ile Thr Gly Leu 35 40 45Phe Leu Gln His Pro Glu Thr Thr Val Ala Ile Thr Gly Thr Asp Ile 50 55 60Val Phe Thr Ala Lys Val Asp Ser Thr Thr Leu Ser Arg Lys Pro Thr65 70 75 80Ile Lys Trp Leu Lys Gly Lys Trp Leu Asp Leu Gly Ser Lys Ala Gly 85 90 95Lys His Met Gln Phe Lys Glu Thr Phe Asp Arg Ala Thr Lys Ile Tyr 100 105 110Thr Trp Asp Met Lys Ile Ile Lys Val Val Pro Gly Asp Ala Gly Ala 115 120 125Tyr Arg Cys Glu Val Thr Ser Lys Asp Lys Cys Asp Ser Ser Ala Phe 130 135 140Asp Ile Ser Val Glu Ala Val Gln Ile Glu Glu Glu His Asp Ser Leu145 150 155 160Ala Ala Phe Lys Arg Thr Asp Ala Gly Glu Asp Glu Gly Ser Leu Asp 165 170 175Phe Ser Ala Leu Leu Lys Ala Thr Lys Lys Lys Lys Lys Pro Val Val 180 185 190Asp Glu Glu Lys Ala Asp Val Trp Glu Ile Leu Lys Asn Ala Gln Pro 195 200 205Ser Glu Tyr Glu Lys Ile Ala Phe Glu Tyr Gly Ile Thr Asp Leu Arg 210 215 220Gly Leu Leu Lys Arg Leu Lys Lys Met Lys Thr Val Glu Pro Lys His225 230 235 240Ser Asp Ala Phe Leu Lys Arg Met Glu Ser Ala Tyr Ser Val Asp Lys 245 250 255Gly Lys Lys Ile Val Leu Gln Val Glu Val Val Asp Pro Asn Ala Gln 260 265 270Val Lys Trp Leu Lys Asn Gly Gln Glu Ile Lys Ser Ser Ala Lys Tyr 275 280 285Ile Ile Glu Ser Val Gly Asn Ile Arg Thr Leu Thr Ile Asn Lys Cys 290 295 300Ser Leu Ala Asp Asp Ala Ala Tyr Glu Cys Val Ile Gly Glu Glu Lys305 310 315 320Ser Phe Thr Glu Val Phe Val Lys Glu Pro Pro Val Thr Ile Thr Lys 325 330 335Leu Leu Asp Asp Tyr His Val Val Val Gly Glu Arg Val Glu Phe Glu 340 345 350Val Glu Val Ser Val Glu Gly Ala His Val Asn

Trp Met Phe Glu Asp 355 360 365Gln Glu Leu Ser Arg Asp Thr His Lys Tyr Arg Phe Lys Lys Asp Gly 370 375 380Leu Lys His Met Leu Ile Ile Gln Glu Ala Thr Leu Asp Asp Ile Gly385 390 395 400Met Tyr Trp Cys Phe Thr Asn Gly Gly Arg Thr Lys Gly Glu Leu Glu 405 410 415Val Glu Glu Lys Glu Leu Glu Val Leu Gln Asn Ile Ala Asp Leu Thr 420 425 430Val Lys Ala Glu Asp Gln Ala Met Phe Lys Cys Glu Val Ser Asp Glu 435 440 445Lys Val Thr Gly Lys Trp Leu Lys Asp Gly Val Glu Val Leu Pro Ser 450 455 460Ser Arg Ile Lys Leu Thr His Ile Gly Arg Ile His Arg Leu Thr Ile465 470 475 480Asp Asp Val Lys Pro Glu Asp Ala Gly Asp Tyr Thr Phe Ile Pro Asp 485 490 495Gly Tyr Ala Leu Ser Leu Ser Ala Lys Leu Asn Phe Leu Glu Ile Lys 500 505 510Ile Asp Tyr Val Pro Arg Gln Glu Pro Pro Lys Ile His Leu Asp Thr 515 520 525Thr Gly Asn Met Val Ser Gln Asn Thr Ile Ile Val Val Ala Gly Asn 530 535 540Lys Leu Arg Leu Asp Val Glu Ile Ser Gly Glu Pro Pro Pro Thr Val545 550 555 560Val Trp Ala Lys Gly Asp Thr Ala Ile Thr Ala Val Glu Gly Arg Val 565 570 575Arg Thr Glu Ser Arg Lys Asp Leu Ser Cys Phe Val Ile Glu Gly Ala 580 585 590Glu Arg Glu Asp Glu Gly Asn Tyr Thr Ile Ile Val Thr Asn Pro Ala 595 600 605Gly Glu Asp Lys Ala His Leu Phe Val Lys Ile Val Asp Val Pro Asp 610 615 620Cys Pro Glu Asn Val Lys Cys Thr Ser Val Gly Glu Asp Cys Ala Thr625 630 635 640Met Val Trp Asp Pro Pro Lys Phe Asp Gly Gly Ala Pro Val Thr Gly 645 650 655Tyr Leu Met Glu Arg Lys Lys Lys Gly Ser Thr Arg Trp Thr Lys Leu 660 665 670Asn Phe Asp Val Tyr Glu Gly Val Thr Tyr Glu Ala Lys Arg Met Ile 675 680 685Glu Gly Val Leu Tyr Glu Met Arg Val Tyr Ala Val Asn Gly Ile Gly 690 695 700Met Ser Gln Pro Ser Leu Asn Ser Lys Pro Phe Met Pro Ile Ala Ala705 710 715 720Thr Ser Glu Pro Leu Gly Leu Lys Val His Asp Val Thr Asp Thr Thr 725 730 735Cys Thr Leu Lys Trp Leu Ala Pro Glu Lys Ile Gly Ala Gly Gly Leu 740 745 750Asp Gly Tyr Val Ile Glu Tyr Cys Lys Glu Gly Asp Thr Glu Trp Val 755 760 765Val Ala Asn Thr Glu Leu Val Glu Arg Gln Asn Tyr Thr Val Arg Asn 770 775 780Leu Pro Thr Ala Glu Lys Ile Asn Phe Arg Val Val Ala Val Asn Ile785 790 795 800Ala Gly Arg Ser Pro Pro Thr Gly Leu Ser Gln Pro Val Thr Ile Arg 805 810 815Glu Ile Val Glu Leu Pro Lys Ile Arg Leu Pro Arg Tyr Leu Arg Gln 820 825 830Lys Tyr Ile Arg Arg Val Gly Asp Lys Ile Asn Leu Thr Ile Pro Phe 835 840 845Gln Gly Lys Pro Arg Pro Lys Val Gln Trp Tyr Lys Asp Gly Glu Glu 850 855 860Leu Asp Thr Arg Val Ala Ser Ile Arg Asn Ser Glu Val Asp Ser Ile865 870 875 880Leu Phe Ile Arg Ser Ala Glu Arg Ser His Ser Gly Lys Tyr Glu Leu 885 890 895Val Leu Gln Ile Glu Asn Met Glu Ala Arg Ala Ile Leu Glu Ile Arg 900 905 910Ile Val Glu Ala Pro Gly Pro Pro Glu Val Val Lys Val Thr Asp Val 915 920 925Trp Gly Phe Asn Ala Ala Leu Glu Trp Lys Pro Pro Lys Asp Asp Gly 930 935 940Asn Cys Glu Ile Thr Gly Tyr Thr Ile Gln Lys Ala Asp Lys Lys Thr945 950 955 960Asn Glu Trp Phe Thr Ile Tyr Glu His Asn Arg Arg Thr Asn Cys Thr 965 970 975Ala Ser Asp Leu Ile Met Gly Asn Glu Tyr Met Phe Arg Val Phe Ser 980 985 990Glu Asn Leu Val Gly Lys Ser Glu Asp Phe Cys Leu Ser Lys Asp Thr 995 1000 1005Ala Ile Ile Pro Lys Ile Gly Leu Glu Tyr Asn Pro Pro Pro Phe 1010 1015 1020Lys Glu Lys Asp Met Gln Ser Ala Pro Lys Phe Ile Gln Pro Leu 1025 1030 1035Leu Asp Arg Ser Val Val Ala Gly Tyr Ser Thr Thr Ile Ser Ser 1040 1045 1050Ala Val Lys Thr Cys Pro Lys Pro Lys Ile Arg Trp Leu Arg Asn 1055 1060 1065Lys Ile Pro Leu Asp Glu Asn Pro Arg Phe Leu Met Gln Asn Asn 1070 1075 1080Gln Gly Val Leu Thr Leu Asn Ile Arg Lys Pro Ser Gln Tyr Asp 1085 1090 1095Gly Gly Lys Phe Thr Cys Lys Ala Ile Asn Ser Leu Gly Glu Asp 1100 1105 1110Val Val Glu Cys Thr Leu Leu Val Arg Ala Leu Lys Asp Lys Glu 1115 1120 1125Asp Gly Asp Glu Lys 11301228PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 122Ala Asp Val Trp Glu Ile Leu Lys1 512314PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 123Glu Leu Glu Val Leu Gln Asn Ile Ala Asp Leu Thr Val Lys1 5 101248PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 124Glu Pro Pro Val Thr Ile Thr Lys1 51258PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 125Gly Glu Leu Glu Val Glu Glu Lys1 51267PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 126His Ser Asp Ala Phe Leu Lys1 512715PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 127Ile Ala Phe Glu Tyr Gly Ile Thr Asp Leu Arg Gly Leu Leu Lys1 5 10 1512811PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 128Ile Gly Leu Glu Tyr Asn Pro Pro Pro Phe Lys1 5 1012914PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 129Ile Asn Leu Thr Ile Pro Phe Gln Gly Lys Pro Arg Pro Lys1 5 1013015PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 130Ile Val Leu Gln Val Glu Val Val Asp Pro Asn Ala Gln Val Lys1 5 10 1513114PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 131Leu Asn Phe Asp Val Tyr Glu Gly Val Thr Tyr Glu Ala Lys1 5 101327PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 132Leu Asn Phe Leu Glu Ile Lys1 51339PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 133Asn Ala Gln Pro Ser Glu Tyr Glu Lys1 513418PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 134Arg Thr Asp Ala Gly Glu Asp Glu Gly Ser Leu Asp Phe Ser Ala Leu1 5 10 15Leu Lys1358PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 135Ser Phe Thr Glu Val Phe Val Lys1 513613PRTArtificial Sequencemyosin-binding protein C, fast-type-like fragment 136Val Asp Ser Thr Thr Leu Ser Arg Lys Pro Thr Ile Lys1 5 101371551DNAArtificial SequenceATP synthase subunit beta, mitochondrial 137atgttaggag ctgtgggacg ctgctgcacc ggggctctcc aggcacttaa gcctggggtc 60cagcccctga aggcccttgt aggatcgcca tctgtccttg gacgcagaga ctactctgca 120cctgctgctg ctgccgcctt cgcccatggc aggatcgtag cggtcatcgg tgccgtcgtc 180gacgtccagt tcgatgaggg cctcccaccc atcctcaatg ccctggaagt tgctgggcgt 240gagtccaggc tagttctgga ggtggcacag catcttggtg agaacactgt gcgtaccatt 300gccatggatg gtactgaagg tcttgtccgt ggacagaagg ttgtggacac tggcgatccc 360atcagaatcc cagtgggtcc cgagacccta ggcagaatca tgaatgtcat tggagagccc 420attgatgaga gggggccaat ctccaccaag cagactgccg ccatccacgc tgaggcccca 480gagttcactg acatgagtgt ggagcaggag atcctggtaa ctggcatcaa ggtggtagat 540ctgctggctc cctatgccaa gggaggcaaa atcggtctgt tcggtggtgc tggtgtgggc 600aagactgtgt tgatcatgga gctgatcaac aatgtggcca aggcccatgg tggttactct 660gtgtttgccg gagtgggaga gcgtacccgc gagggaaatg acttgtacca cgagatgatt 720gagtcgggtg tcatcaacct gaaggatgac acctccaagg tggcgctggt gtacggacaa 780atgaacgagc ccccaggcgc ccgtgcccgt gtggctctga ctggtctgac cgtggcagag 840tatttccgtg accaggaggg tcaggatgtg ctgctcttca tcgataacat cttccgtttc 900acccaggctg gctccgaggt gtctgccctg ctgggtcgta tcccctccgc tgtgggttac 960cagcccaccc tggccactga catgggtacc atgcaggaga gaatcaccac caccaagaag 1020ggttccatca cctctgtgca ggccatctat gtgccggctg acgatttgac tgatcccgcc 1080cctgccacca ccttcgctca cttggacgcc accactgtgc tgtcccgtgc aatcgctgag 1140ctgggtatct accctgctgt ggatcccctg gattccacct cccgtatcat ggaccccaac 1200attgtcggcg ctgagcacta tgatgttgct cgtggcgtgc agaagatcct ccaggactac 1260aagtccctgc aggatatcat tgccatcctg ggtatggatg agttgtctga ggaggacaag 1320ctgattgtct ctcgcgcccg caagatccag cgtttcctgt cccagccctt ccaggtggcg 1380gaggtattca ccggtcatgc tggcaagctg gtgccactca aggagaccat cagtggcttc 1440cagagcattc taaatggtga gtacgatgcc ctgcccgagc aggctttcta catggttgga 1500cccatcgagg aggtggttga gaaggctgca cagatggcca aggatctcgc a 1551138517PRTArtificial SequenceATP synthase subunit beta, mitochondrial 138Met Leu Gly Ala Val Gly Arg Cys Cys Thr Gly Ala Leu Gln Ala Leu1 5 10 15Lys Pro Gly Val Gln Pro Leu Lys Ala Leu Val Gly Ser Pro Ser Val 20 25 30Leu Gly Arg Arg Asp Tyr Ser Ala Pro Ala Ala Ala Ala Ala Phe Ala 35 40 45His Gly Arg Ile Val Ala Val Ile Gly Ala Val Val Asp Val Gln Phe 50 55 60Asp Glu Gly Leu Pro Pro Ile Leu Asn Ala Leu Glu Val Ala Gly Arg65 70 75 80Glu Ser Arg Leu Val Leu Glu Val Ala Gln His Leu Gly Glu Asn Thr 85 90 95Val Arg Thr Ile Ala Met Asp Gly Thr Glu Gly Leu Val Arg Gly Gln 100 105 110Lys Val Val Asp Thr Gly Asp Pro Ile Arg Ile Pro Val Gly Pro Glu 115 120 125Thr Leu Gly Arg Ile Met Asn Val Ile Gly Glu Pro Ile Asp Glu Arg 130 135 140Gly Pro Ile Ser Thr Lys Gln Thr Ala Ala Ile His Ala Glu Ala Pro145 150 155 160Glu Phe Thr Asp Met Ser Val Glu Gln Glu Ile Leu Val Thr Gly Ile 165 170 175Lys Val Val Asp Leu Leu Ala Pro Tyr Ala Lys Gly Gly Lys Ile Gly 180 185 190Leu Phe Gly Gly Ala Gly Val Gly Lys Thr Val Leu Ile Met Glu Leu 195 200 205Ile Asn Asn Val Ala Lys Ala His Gly Gly Tyr Ser Val Phe Ala Gly 210 215 220Val Gly Glu Arg Thr Arg Glu Gly Asn Asp Leu Tyr His Glu Met Ile225 230 235 240Glu Ser Gly Val Ile Asn Leu Lys Asp Asp Thr Ser Lys Val Ala Leu 245 250 255Val Tyr Gly Gln Met Asn Glu Pro Pro Gly Ala Arg Ala Arg Val Ala 260 265 270Leu Thr Gly Leu Thr Val Ala Glu Tyr Phe Arg Asp Gln Glu Gly Gln 275 280 285Asp Val Leu Leu Phe Ile Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly 290 295 300Ser Glu Val Ser Ala Leu Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr305 310 315 320Gln Pro Thr Leu Ala Thr Asp Met Gly Thr Met Gln Glu Arg Ile Thr 325 330 335Thr Thr Lys Lys Gly Ser Ile Thr Ser Val Gln Ala Ile Tyr Val Pro 340 345 350Ala Asp Asp Leu Thr Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu 355 360 365Asp Ala Thr Thr Val Leu Ser Arg Ala Ile Ala Glu Leu Gly Ile Tyr 370 375 380Pro Ala Val Asp Pro Leu Asp Ser Thr Ser Arg Ile Met Asp Pro Asn385 390 395 400Ile Val Gly Ala Glu His Tyr Asp Val Ala Arg Gly Val Gln Lys Ile 405 410 415Leu Gln Asp Tyr Lys Ser Leu Gln Asp Ile Ile Ala Ile Leu Gly Met 420 425 430Asp Glu Leu Ser Glu Glu Asp Lys Leu Ile Val Ser Arg Ala Arg Lys 435 440 445Ile Gln Arg Phe Leu Ser Gln Pro Phe Gln Val Ala Glu Val Phe Thr 450 455 460Gly His Ala Gly Lys Leu Val Pro Leu Lys Glu Thr Ile Ser Gly Phe465 470 475 480Gln Ser Ile Leu Asn Gly Glu Tyr Asp Ala Leu Pro Glu Gln Ala Phe 485 490 495Tyr Met Val Gly Pro Ile Glu Glu Val Val Glu Lys Ala Ala Gln Met 500 505 510Ala Lys Asp Leu Ala 51513911PRTArtificial SequenceATP synthase subunit beta, mitochondrial fragment 139Ile Gly Leu Phe Gly Gly Ala Gly Val Gly Lys1 5 1014021PRTArtificial SequenceATP synthase subunit beta, mitochondrial fragment 140Ile Gln Arg Phe Leu Ser Gln Pro Phe Gln Val Ala Glu Val Phe Thr1 5 10 15Gly His Ala Gly Lys 2014113PRTArtificial SequenceATP synthase subunit beta, mitochondrial fragment 141Thr Val Leu Ile Met Glu Leu Ile Asn Asn Val Ala Lys1 5 1014210PRTArtificial SequenceATP synthase subunit beta, mitochondrial fragment 142Val Val Asp Leu Leu Ala Pro Tyr Ala Lys1 5 10143996DNAArtificial SequenceL-lactate dehydrogenase A chain-like 143atgactacca aggagaagct gatcacccat gtgttggctg gtgagcctgt tggctcccgg 60agcaaggtga cagttgttgg cgtcggcatg gttggcatgg cctccgcagt cagcgtcctg 120ctcaaggacc tgtgcgatga gctgtgcctg attgacgtga tggaagataa actgaagggt 180gaggtcatgg acctgcagca tggcagcctc ttctgcaaga ctcacaagat tgtgggcgac 240aaggactaca gtacgactgc ccactccaag gtggtggtgg tcacagccgg tgctcgtcag 300caagagggtg agagccgtct gaacctggtg cagcgtaacg tcaacatctt caaattcata 360attccccaga tcgtcaagta cagccccaac gccatcctgc tggtcgtctc caatcctgtt 420gacatcctaa cctacgtggc ttggaagctg agtggtttcc cccgtcaccg cgtcatcggt 480tccggcacca acctggactc tggtcgtttc cgccacctga tgggcgagaa gctacacctt 540cacccatcca gctgtcacgg ctggatcatt ggagaacacg gagactccag cgtgcccgta 600tggagcggtg tgaatgttgc cggtgtttcc ctgaagggcc tgaacccaga catgggcaca 660gacgcagaca aggaggactg gaagcacgtc cacaagatgg tggtcgacgg tgcctatgag 720gtcatcaagc tgaagggtta cacctcctgg gctatcggca tgtccgtcgc tgacctggtt 780gagagcatcc tgaagaacct ccacaaagtc caccctgtgt ccaccctggt caagggaatg 840cacggtgtga aggaggaggt gtttctcagc gtgccctgcg tgctgggaaa cagcggtctg 900accgacgtca tccacatgac tctgaagccc gaggaggaga agcagctgag caacagtgcc 960gagaccctat ggggcgtaca gaaagagctc accttg 996144332PRTArtificial SequenceL-lactate dehydrogenase A chain-like 144Met Thr Thr Lys Glu Lys Leu Ile Thr His Val Leu Ala Gly Glu Pro1 5 10 15Val Gly Ser Arg Ser Lys Val Thr Val Val Gly Val Gly Met Val Gly 20 25 30Met Ala Ser Ala Val Ser Val Leu Leu Lys Asp Leu Cys Asp Glu Leu 35 40 45Cys Leu Ile Asp Val Met Glu Asp Lys Leu Lys Gly Glu Val Met Asp 50 55 60Leu Gln His Gly Ser Leu Phe Cys Lys Thr His Lys Ile Val Gly Asp65 70 75 80Lys Asp Tyr Ser Thr Thr Ala His Ser Lys Val Val Val Val Thr Ala 85 90 95Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg 100 105 110Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Gln Ile Val Lys Tyr Ser 115 120 125Pro Asn Ala Ile Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140Tyr Val Ala Trp Lys Leu Ser Gly Phe Pro Arg His Arg Val Ile Gly145 150 155 160Ser Gly Thr Asn Leu Asp Ser Gly Arg Phe Arg His Leu Met Gly Glu 165 170 175Lys Leu His Leu His Pro Ser Ser Cys His Gly Trp Ile Ile Gly Glu 180 185 190His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Val Asn Val Ala Gly 195 200 205Val Ser Leu Lys Gly Leu Asn Pro Asp Met Gly Thr Asp Ala Asp Lys 210 215 220Glu Asp Trp Lys His Val His Lys Met Val Val Asp Gly Ala Tyr Glu225 230 235 240Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Met Ser Val 245 250 255Ala Asp Leu Val Glu Ser Ile Leu Lys Asn Leu His Lys Val His Pro 260 265 270Val Ser Thr Leu Val Lys Gly Met His Gly

Val Lys Glu Glu Val Phe 275 280 285Leu Ser Val Pro Cys Val Leu Gly Asn Ser Gly Leu Thr Asp Val Ile 290 295 300His Met Thr Leu Lys Pro Glu Glu Glu Lys Gln Leu Ser Asn Ser Ala305 310 315 320Glu Thr Leu Trp Gly Val Gln Lys Glu Leu Thr Leu 325 3301458PRTArtificial SequenceL-lactate dehydrogenase A chain-like fragment 145Phe Ile Ile Pro Gln Ile Val Lys1 514616PRTArtificial SequenceL-lactate dehydrogenase A chain-like fragment 146Leu Ile Thr His Val Leu Ala Gly Glu Pro Val Gly Ser Arg Ser Lys1 5 10 1514714PRTArtificial SequenceL-lactate dehydrogenase A chain-like fragment 147Thr His Val Leu Ala Gly Glu Pro Val Gly Ser Arg Ser Lys1 5 101489PRTArtificial SequenceL-lactate dehydrogenase A chain-like fragment 148Val His Pro Val Ser Thr Leu Val Lys1 514928PRTArtificial SequenceL-lactate dehydrogenase A chain-like fragment 149Val Val Val Val Thr Ala Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg1 5 10 15Leu Asn Leu Val Gln Arg Asn Val Asn Ile Phe Lys 20 25

Claims

1. A kit for diagnosing a fish allergy, the kit comprising, as an antigen, at least one protein defined in any one of the following (1) to (9): (1) (1A) Alpha-actinin-3 or a variant thereof, which is a protein defined below in any of (1A-a) to (1A-e) which is an antigen for the fish allergy: (1A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 2; (1A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2; (1A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 1; (1A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 1; or (1A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1; or (1B) a protein comprising an amino acid sequence of SEQ ID NO: 2 or 3; (2) (2A) EEF1A2 binding protein-like or a variant thereof, which is a protein defined below in any of (2A-a) to (2A-e) which is an antigen for the fish allergy: (2A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 5; (2A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 5; (2A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 4; (2A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 4; or (2A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 4; or (2B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8; (3) (3A) Alpha-1,4-glucan phosphorylase or a variant thereof, which is a protein defined below in any of (3A-a) to (3A-e) which is an antigen for the fish allergy: (3A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 10; (3A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 10; (3A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 9; (3A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 9; or (3A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 9; or (3B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 10-16; (4) (4A) Elongation factor 2 or a variant thereof, which is a protein defined below in any of (4A-a) to (4A-e) which is an antigen for the fish allergy: (4A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 19; (4A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 19; (4A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 18; (4A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 18; or (4A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 18; or (4B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; (5) (5A) Heat shock cognate 70 kDa protein or a variant thereof, which is a protein defined below in any of (5A-a) to (5A-e) which is an antigen for the fish allergy: (5A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 26; (5A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 26; (5A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 25; (5A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 25; or (5A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 25; or (5B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 26-31; (6) (6A) Serotransferrin or a variant thereof, which is a protein defined below in any of (6A-a) to (6A-e) which is an antigen for the fish allergy: (6A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 33; (6A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 33; (6A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 32; (6A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 32; or (6A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 32; or (6B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 33-41; (7) (7A) Myosin binding protein H-like or a variant thereof, which is a protein defined below in any of (7A-a) to (7A-e) which is an antigen for the fish allergy: (7A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 43; (7A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 43; (7A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 42; (7A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 42; or (7A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 42; or (7B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 43-54, (8) (8A) Desmin (Fragment) or a variant thereof, which is a protein defined below in any of (8A-a) to (8A-e) which is an antigen for the fish allergy: (8A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 56; (8A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 56; (8A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 55; (8A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 55; or (8A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 55; or (8B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 56-59; (9) (9A) Capping protein (Actin filament) muscle Z-line beta or a variant thereof, which is a protein defined below in any of (9A-a) to (9A-e) which is an antigen for the fish allergy: (9A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 61; (9A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 61; (9A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 60; (9A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 60; or (9A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 60; or (9B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 61-68.

2. A kit for diagnosing a fish allergy, the kit comprising, as an antigen, at least one protein defined in any one of the following (10) to (14): (10) (10A) Myosin heavy chain, fast skeletal muscle-like or a variant thereof, which is a protein defined below in any of (10A-a) to (10A-e) which is an antigen for the fish allergy: (10A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 70; (10A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 70; (10A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 69; (10A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 69; or (10A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 69; or (10B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 70-108; (11) (11A) Glycogen phosphorylase, muscle form-like or a variant thereof, which is a protein defined below in any of (11A-a) to (11A-e) which is an antigen for the fish allergy: (11A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 110; (11A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 110; (11A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 109; (11A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 109; or (11A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 109; or (11B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 110-119; (12) (12A) Myosin-binding protein C, fast-type-like or a variant thereof, which is a protein defined below in any of (12A-a) to (12A-e) which is an antigen for the fish allergy: (12A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 121; (12A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 121; (12A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 120; (12A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 120; or (12A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 120; or (12B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 121-136; (13) (13A) ATP synthase subunit beta, mitochondrial or a variant thereof, which is a protein defined below in any of (13A-a) to (13A-e) which is an antigen for the fish allergy: (13A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 138; (13A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 138; (13A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 137; (13A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 137; or (13A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 137; or (13B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 138-142; (14) (14A) L-lactate dehydrogenase A chain-like or a variant thereof, which is a protein defined below in any of (14A-a) to (14A-e) which is an antigen for the fish allergy: (14A-a) a protein comprising an amino acid sequence with deletion, substitution, insertion or addition of one or several amino acids in SEQ ID NO: 144; (14A-b) a protein comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 144; (14A-c) a protein comprising an amino acid sequence encoded by a nucleotide sequence with deletion, substitution, insertion or addition of one or several nucleotides in SEQ ID NO: 143; (14A-d) a protein comprising an amino acid sequence encoded by a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 143; or (14A-e) a protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 143; or (14B) a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 144-149.

3. A composition for diagnosing a fish allergy, the composition comprising, as an antigen, at least one of proteins as defined in any of (1) to (9) of claim 1.

4. A method for providing an indicator for diagnosing a fish allergy in a subject, the method comprising the steps of: (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody; (ii) detecting binding between the IgE antibody present in the sample obtained from the subject and the antigen; and (iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject has a fish allergy is provided; wherein the antigen is at least one of proteins as defined in any of (1) to (9) of claim 1.

5. A pharmaceutical composition comprising at least one of proteins as defined in any of (1) to (9) of claim 1.

6. The pharmaceutical composition according to claim 5 for the treatment of an allergy to a fish.

7. A fish or a processed product of fish in which an antigen is eliminated or reduced, wherein the antigen is at least one of proteins as defined in any of (1) to (9) of claim 1.

8. A tester for determining the presence or absence of a fish antigen in an object of interest, the tester comprising an antibody that binds to at least one of proteins as defined in any of (1) to (9) of claim 1.

9. A tester for determining the presence or absence of an antigen causing an allergy to fish in an object of interest, the tester comprising a primer having a nucleotide sequence complementary to at least one part of one of the nucleotide sequences set forth in SEQ ID NO: 1, 4, 9, 18, 25, 32, 42, 55, 60, 69, 109, 120, 137, or 143.

10. A fish-derived antigen, which is at least one of proteins as defined in any of (1) to (9) of claim 1 and causes an allergy to a fish.

11. A composition for diagnosing a fish allergy, the composition comprising, as an antigen, at least one of proteins as defined in any of (10) to (14) of claim 2.

12. A method for providing an indicator for diagnosing a fish allergy in a subject, the method comprising the steps of: (i) contacting a sample obtained from the subject with an antigen, wherein the sample is a solution comprising an IgE antibody; (ii) detecting binding between the IgE antibody present in the sample obtained from the subject and the antigen; and (iii) when the binding between the IgE antibody in the subject and the antigen is detected, an indicator of the fact that the subject has a fish allergy is provided; wherein the antigen is at least one of proteins as defined in any of (10) to (14) of claim 2.

13. A pharmaceutical composition comprising at least one of proteins as defined in any of (10) to (14) of claim 2.

14. The pharmaceutical composition according to claim 13 for the treatment of an allergy to a fish.

15. A fish or a processed product of fish in which an antigen is eliminated or reduced, wherein the antigen is at least one of proteins as defined in any of (10) to (14) of claim 2.

16. A tester for determining the presence or absence of a fish antigen in an object of interest, the tester comprising an antibody that binds to at least one of proteins as defined in any of (10) to (14) of claim 2.

17. A fish-derived antigen, which is at least one of proteins as defined in any of (10) to (14) of claim 2 and causes an allergy to a fish.

Images